Methods for attenuating virus strains for diagnostic and therapeutic uses

ABSTRACT

Modified or attenuated viruses and methods for preparing the modified viruses and modulating attenuation are provided. Vaccines that contain the viruses are provided. The viruses can be used in methods of treatment of diseases, such as proliferative and inflammatory disorders, including as anti-tumor agents. The viruses also can be used in diagnostic methods.

RELATED APPLICATIONS

Benefit of priority is claimed under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 60/852,390, to Nanhai Chen, Aladar A.Szalay, Yong A. Yu and Qian Zhang, filed on Oct. 16, 2006, entitled“MODIFIED VACCINIA VIRUS STRAINS FOR USE IN DIAGNOSTIC AND THERAPEUTICMETHODS,” to U.S. Provisional Application Ser. No. 60/933,050, to QianZhang and Nanhai Chen, filed on Jun. 4, 2007, entitled “VECTOR FORVACCINIA VIRUS,” to U.S. Provisional Application Ser. No. 60/950,587, toNanhai Chen and Yong A. Yu, filed on Jul. 18, 2007, entitled “USE OFMODIFIED VACCINIA VIRUS STRAINS IN COMBINATION WITH A CHEMOTHERAPEUTICAGENT FOR USE IN THERAPEUTIC METHODS,” and to U.S. ProvisionalApplication Ser. No. 60/994,794, to Alexa Frentzen, Nanhai Chen, QianZhang, Yong A. Yu and Aladar A. Szalay, filed on Sep. 21, 2007, entitled“MODIFIED VACCINIA VIRUS STRAINS.” The subject matter of each of theseapplications is incorporated by reference in its entirety.

This application is related to U.S. Application No. (Attorney Dkt. No.17248-016001/4816) to Nanhai Chen, Alexa Frentzen, Aladar A. Szalay,Yong A. Yu and Qian Zhang, filed on Oct. 16, 2007, entitled “MODIFIEDVACCINIA VIRUS STRAINS FOR USE IN DIAGNOSTIC AND THERAPEUTIC METHODS,”which also claims priority to U.S. Provisional Application Ser. No.60/852,390, U.S. Provisional Application Ser. No. 60/933,050, U.S.Provisional Application Ser. No. 60/950,587, and to U.S. ProvisionalApplication Ser. No. 60/994,794.

This application is related to International Application No. (AttorneyDkt. No. 17248-015WO1/4815PC) to Nanhai Chen, Alexa Frentzen, Aladar A.Szalay, Yong A. Yu and Qian Zhang, filed on Oct. 16, 2007, entitled“MODIFIED VACCINIA VIRUS STRAINS FOR USE IN DIAGNOSTIC AND THERAPEUTICMETHODS,” which also claims priority to U.S. Provisional ApplicationSer. No. 60/852,390, U.S. Provisional Application Ser. No. 60/933,050,U.S. Provisional Application Ser. No. 60/950,587, and to U.S.Provisional Application Ser. No. 60/994,794.

This application is related to U.S. application Ser. No. 10/872,156, toAladar A. Szalay, Tatyana Timiryasova, Yong A. Yu and Qian Zhang, filedon Jun. 18, 2004, entitled “MICROORGANISMS FOR THERAPY,” which claimsthe benefit of priority under 35 U.S.C. §19(a) to each of EP 03 013826.7, filed 18 Jun. 2003, entitled “Recombinant vaccinia viruses usefulas tumor-specific delivery vehicle for cancer gene therapy andvaccination,” EP 03 018 478.2, filed 14 Aug. 2003, entitled “Method forthe production of a polypeptide, RNA or other compound in tumor tissue,”and EP 03 024 283.8, filed 22 Oct. 2003, entitled “Use of aMicroorganism or Cell to Induce Autoimmunization of an Organism Againsta Tumor.”

This application also is related to International Application Serial No.PCT/US04/19866, filed on Jun. 18, 2004. This application also is relatedto U.S. application Ser. No. 10/866,606, filed Jun. 10, 2004, entitled“Light emitting microorganisms and cells for diagnosis and therapy oftumors,” which is a continuation of U.S. application Ser. No.10/189,918, filed Jul. 3, 2002, U.S. Application filed May 19, 2004 Ser.No. 10/849,664, entitled, “Light emitting microorganisms and cells fordiagnosis and therapy of diseases associated with wounded or inflamedtissue” which is a continuation of U.S. application Ser. No. 10/163,763,filed Jun. 5, 2003, International PCT Application WO 03/014380, filedJul. 31, 2002, entitled “Microorganisms and Cells for Diagnosis andTherapy of Tumors,” PCT Application WO 03/104485, filed Jun. 5, 2003,entitled, “Light Emitting Microorganisms and Cells for Diagnosis andTherapy of Diseases Associated with Wounded or Inflamed tissue,” EPApplication No. 01 118 417.3, filed Jul. 31, 2001, entitled“Light-emitting microorganisms and cells for tumor diagnosis/therapy,”EP Application No. 01 125 911.6, filed Oct. 30, 2001, entitled “Lightemitting microorganisms and cells for diagnosis and therapy of tumors,”EP Application No. 02 0794 632.6, filed Jan. 28, 2004, entitled“Microorganisms and Cells for Diagnosis and Therapy of Tumors,” and EPApplication No. 02 012 552.2, filed Jun. 5, 2002, entitled “LightEmitting Microorganisms and Cells for Diagnosis and Therapy of Diseasesassociated with wounded or inflamed tissue.”

This application also is related to U.S. application Ser. No.11/827,518, to Jochen Stritzker, Phil Hill, Aladar A. Szalay, Yong A. Yuand Qian Zhang, entitled “METHODS AND COMPOSITIONS FOR DETECTION OFMICROORGANISMS AND CELLS AND TREATMENT OF DISEASES AND DISORDERS,” filedJul. 11, 2007.

The subject matter of each of the applications mentioned above isincorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF A SEQUENCE LISTING PROVIDED ON COMPACTDISCS

An electronic version on compact disc (CD-R) of the Sequence Listing isfiled herewith in duplicate (labeled Copy #1 and Copy #2), the contentsof which are incorporated by reference in their entirety. Thecomputer-readable file on each of the aforementioned compact discs,created on Oct. 16, 2007, is identical, 854 kilobytes in size, andentitled 4815SEQ.001.txt.

FIELD OF THE INVENTION

Modified and/or attenuated viruses and methods for preparing themodified viruses and modulating attenuation are provided. Diagnostic andtherapeutic methods also are provided.

BACKGROUND

Viruses for therapeutic and diagnostic methods often are pathogenic andmust be attenuated to increase their safety for administration.Attenuation can be effected by repeated passage through cell linesand/or through animals to screen for strains that have reducedpathogenicity. Other methods for attenuation of a virus involveproduction of recombinant viruses that have a modification in one ormore viral genes that results in loss or reduced expression of a viralgene or inactivation of a viral protein. Once attenuated viruses aregenerated, methods for increasing the attenuation of the virus ofteninvolve selecting or identifying additional genes for mutation,combining mutations and/or insertion of heterologous genes forexpression of proteins that alter the in vivo pathogenicity of the virus(see e.g., U.S. Pat. No. 6,265,189 and U.S. Patent Publication No.2006-0099224). The effects of combinations of modifications, however,are difficult to predict and require extensive testing to determine whatcombinations of modifications yields a desired level of attenuation.Further complicating the process is the fact that mutations oftendecrease or abolish viral functions that are required for viralreplication or life cycle progression. Essential viral functions oftenare provided in trans in order to produce the mature virions forinfection (see e.g., U.S. Pat. Nos. 5,750,396, 6,261,551, 6,924,128,6,974,695). Thus, packaging cell lines that express the essential viralproteins are required for viral propagation. Such cell lines, however,can be challenging to generate due to the toxicity of the viral proteinsthat are expressed.

Mutation of non-essential genes is a method of attenuation thatpreserves the ability of the virus to propagate without the need of apackaging cell lines. In viruses such as vaccinia virus, mutations innon-essential genes, such as the thymidine kinase (TK) gene orhemagglutinin (HA) gene have been employed to attenuate the virus (e.g.,Buller et al. (1985) Nature 317, 813-815, Shida et al. (1988) J. Virol.62(12):4474-80, Taylor et al. (1991) J. Gen. Virol. 72 (Pt 1):125-30,U.S. Pat. Nos. 5,364,773, 6,265,189, 7,045,313). The inactivation ofthese genes decreases the overall pathogenicity of the virus withouteliminating the ability of the viruses to replicate in certain celltypes. Further modulation of the attenuation of the virus similarly isdifficult, since it can require identification of additionalnon-essential genes for modification, followed by testing ofcombinations of mutations in order to select a recombinant virus with adesired level of attenuation.

In view of the efforts to generate attenuated viruses for therapy,including the methods mentioned above, there still exists a need formethods for attenuating viruses. Accordingly, it is among the objectsherein, to provide methods for attenuating viruses and to provideattenuated viruses and diagnostic and/or therapeutic methods that employsuch viruses.

SUMMARY

Provided herein are methods for attenuating viruses. The viruses can beused in therapeutic and diagnostic methods. Also provided are attenuatedviruses.

The methods for attenuation permit modulation of the levels of viralattenuation without the need to mutate restrictive combinations of viralgenes or provide additional therapeutic genes for in vivo attenuation.The methods permit modulation of the attenuation of the virus in apredictable manner. Provided are methods for systematically altering avirus to a level of attenuation that is desired for a particularapplication of the virus. Also provided are attenuated viruses.

Therapeutic viruses also are provided. The viruses can be used astherapeutics. In addition they can be employed as starting materials inthe methods for modulating attenuation. The therapeutic viruses cancontain a heterologous nucleic acid, inserted for its encoded protein orfor attenuation. The heterologous nucleic acid can contain an openreading frame operably linked to a promoter. The heterologous nucleicacid can be operatively linked to a native promoter or a heterologous(with respect to the virus) promoter. Any suitable promoters, includingsynthetic and naturally-occurring and modified promoters, can be used.Exemplary promoters include synthetic promoters, including syntheticviral and animal promoters. Native promoter or heterologous promotersinclude, but are not limited to, viral promoters, such as vaccinia virusand adenovirus promoters. Vaccinia viral promoters can be synthetic ornatural promoters, and include vaccinia early, intermediate, early/lateand late promoters. Exemplary vaccinia viral promoters for use in themethods can include, but are not limited to, P_(7.5k), P_(11k), P_(SE),P_(SEL), P_(SL), H5R, TK, P28, C11R, G8R, F17R, I3L, I8R, A1L, A2L, A3L,H1L, H3L, H5L, H6R, H8R, D1R, D4R, D5R, D9R, D11L, D12L, D13L, M1L, N2L,P4b or K1 promoters. Other viral promoters can include, but are notlimited to, adenovirus late promoter, Cowpox ATI promoter, or T7promoter.

Methods provided herein for modification of viruses, particularlytherapeutic viruses, include steps of addition, deletion and/ormodification of a heterologous nucleic acid in the viral genome. Suchmodifications of viruses result in altering the level of attenuation ofthe virus compared to the unmodified virus. Methods provided herein forthe modulation of attenuation of a therapeutic virus can increase ordecrease the level of attenuation of the therapeutic virus compared toan unmodified therapeutic virus.

Provided herein are methods to alter the attenuation of a therapeuticvirus where a promoter contained in the therapeutic virus is modified orreplaced. Such promoters can be replaced by stronger or weakerpromoters, where replacement results in a change in the attenuation ofthe virus. As provided in the methods herein, a promoter contained in atherapeutic virus can be replaced with a natural or synthetic promoter.Exemplary promoters that can replace a promoter contained in atherapeutic virus can be a viral promoter, such as a vaccinia viralpromoter, and can include a vaccinia early, intermediate, early/late orlate promoter. Additional exemplary viral promoters are provided hereinand known in the art and can be used to replace a promoter contained ina therapeutic virus.

Therapeutic viruses for use in the methods provided herein of modulationthe attenuation of the virus can contain a heterologous nucleic acidthat contains an open reading frame that encodes one or more geneproducts. Methods provided herein for modulating the attenuation of atherapeutic virus include modification of a heterologous nucleic acidthat contains an open reading frame. Methods provided herein formodification of the open reading frame can include increasing the lengthof the open reading frame, removal of all or part of the open readingframe or replacement of all or part of the open reading frame.

Provided herein are methods to alter the attenuation of a therapeuticvirus where a heterologous nucleic acid contained in the virus ismodified by removal or all or a portion of the heterologous nucleic acidmolecule. The portion of the heterologous nucleic acid that is removedcan be 1, 2, 3, 4, 5 or more, 10 or more, 15 or more, 20 or more, 50 ormore, 100 or more, 1000 or more, 5000 or more nucleotide bases. Alsoprovided herein are methods to alter the attenuation of a therapeuticvirus where a heterologous nucleic acid contained in the virus ismodified by removal or all or a portion of a first heterologous nucleicacid molecule and replaced by a second heterologous nucleic acidmolecule, where replacement changes the level of attenuation of thevirus. The second heterologous nucleic acid molecule can contain asequence of nucleotides that encodes a protein or can be a non-codingnucleic acid molecule. In some examples, the second heterologous nucleicacid molecule contains an open reading frame operably linked to apromoter. The second heterologous nucleic acid molecule can contain oneor more open reading frames or one or more promoters. Further, the oneor more promoters of the second heterologous nucleic acid molecule canbe one or more stronger promoters or one or more weaker promoters, orcan be a combination or both.

Provided herein are methods for assessing the level of attenuation of atherapeutic virus following addition, deletion and/or modification of aheterologous nucleic acid in the viral genome. Such methods formeasuring the level of attenuation can be performed in vitro or in vivoand can include assessment of changes in any or all of the followingproperties of the virus: a) viral mRNA synthesis, b) viral proteinexpression, c) viral DNA replication, d) viral plaque size, e) viraltiter or f) in vivo toxicity. The methods provided herein can modulatethe attenuation of a therapeutic virus by altering transcription of oneor more viral genes or altering translation of one or more endogenousviral polypeptides during the viral life cycle.

Provided herein are methods for determining the desired level ofattenuation for application of the virus. Exemplary applications of atherapeutic virus include diagnostic applications, therapeuticapplication or a combination thereof. An exemplary therapeuticapplication is treatment of a tumor, cancer or metastasis. An exemplarydiagnostic application is detection of a tumor. The desired level ofattenuation for application of the therapeutic virus can depend on avariety of factors including, but not limited to, the health of asubject prior to administration of the virus to the subject or theselection of the route of administration for the virus.

Provided herein are methods for further modification of therapeuticviruses that have been modified to modulate their attenuation. Includedin such methods are insertion heterologous nucleic acid molecules thatencode a detectable protein or a protein capable of inducing adetectable signal. Exemplary of such proteins are luciferases, such as aclick beetle luciferase, a Renilla luciferase, or a firefly luciferase,fluorescent proteins, such as a GFP or RFP, or proteins that can bind acontrasting agent, chromophore, or a compound or ligand that can bedetected, such as a transferrin receptor or a ferritin. Also included insuch methods are insertion heterologous nucleic acid molecules thatencode a therapeutic gene product, such as a cytokine, a chemokine, animmunomodulatory molecule, a single chain antibody, antisense RNA,siRNA, prodrug converting enzyme, a toxin, an antitumor oligopeptide, ananti-cancer polypeptide antibiotic, angiogenesis inhibitor, or tissuefactor. Such heterologous nucleic acid molecules can be inserted intothe viral genome in an intergenic region or in a locus that encodes anonessential viral gene product, such as hemagglutinin (HA), thymidinekinase (TK), F14.5L, vaccinia growth factor (VGF), A35R, or N1L geneloci. In some examples, methods for further modification of therapeuticviruses, such as vaccinia viruses, that have been modified to modulatetheir attenuation can include replacement of the A34R gene with the A34Rgene from another vaccinia virus strain. For example, in a vaccinia LIVPstrain, the A34R gene can be replaced with the A34R gene from vacciniaIHD-J strain. Such replacement can increase the extracellular envelopedvirus (EEV) form of vaccinia virus or can increase the resistance of thevirus to neutralizing antibodies.

Therapeutic viruses for use in the methods provided herein of modulationthe attenuation of the virus can be, for example, a poxvirus,herpesvirus, adenovirus, adeno-associated virus, lentivirus, retrovirus,rhabdovirus or papillomavirus. Exemplary members of these families ofviruses are vaccinia virus, avipox virus, myxoma virus, cytomegalovirus(CMV), murine Maloney leukemia virus (MMLV), human immunodeficiencyvirus (HIV), and vesicular stomatitis virus (VSV), reovirus, Newcastledisease virus, coxsackievirus, measles virus, influenza virus, mumpsvirus, poliovirus, Seneca valley virus, and semliki forest virus.Exemplary vaccinia virus strains for use in the methods provided hereininclude Western Reserve (WR), Copenhagen, Tashkent, Tian Tan, Lister,Wyeth, IHD-J, and IHD-W, Brighton, Ankara, MVA, Dairen I, LIPV, LC16M8,LC16MO, LIVP, WR 65-16, Connaught, New York City Board of Health.Exemplary LIVP vaccinia viruses provided herein for use in the methodsprovided herein include GLV-1h22, GLV-1h68, GLV-1i69, GLV-1h70,GLV-1h71, GLV-1h72, GLV-1h73, GLV-1h75, GLV-1h81, GLV-1h82, GLV-1h83,GLV-1h84, GLV-1h85, GLV-1h86, GLV-1j87, GLV-1j88, GLV-1j89, GLV-1h90,GLV-1h91, GLV-1h92, GLV-1h96, GLV-1h97, GLV-1h98, GLV-1h104, GLV-1h105,GLV-1h106, GLV-1h107, GLV-1h108 an GLV-1h109.

Provided herein are viruses for use uses therapeutics and/or indiagnostic methods. Exemplary viruses provided herein includerecombinant vaccinia viruses that contain a modified hemagglutinin (HA)gene, thymidine kinase (TK) gene, and F14.5L gene, where one or more ofthe modifications comprises insertion of a heterologous non-codingnucleic acid molecule into the HA gene locus, TK gene locus, or F14.5Lgene locus. In such viruses, a functional HA, TK, and F14.5L polypeptideis not expressed. Exemplary viruses provided herein for therapeutic anddiagnostic use include Lister strain vaccinia viruses, such as GLV-1i69,GLV-1h70, GLV-1h71, GLV-1h72, GLV-1h73, GLV-1h74, GLV-1h81, GLV-1h82,GLV-1h83, GLV-1h84, GLV-1h85, GLV-1h86, GLV-1j87, GLV-1j88, GLV-1j89,GLV-1h90, GLV-1h91, GLV-1h92, GLV-1h96, GLV-1h97, GLV-1h98, GLV-1h104,GLV-1h105, GLV-1h106, GLV-1h107, GLV-1h108 an GLV-1h109.

Viruses provided herein for therapeutic and diagnostic use includerecombinant vaccinia viruses that contain a heterologous nucleic acidmolecule that encodes a therapeutic gene product, such as anangiogenesis inhibitor (e.g., plasminogen kringle 5 domain, anti-VEGFscAb (G6), tTF-RGD, truncated human tissue factor-RGD peptide fusionprotein), a tumor growth suppressor (e.g., IL-24), an immune stimulator(e.g., sIL-6R-IL-6, soluble IL-6 receptor-IL-6 fusion protein).

Such therapeutic gene products can be operably linked to a vacciniapromoter, such as a vaccinia early promoter, a vaccinia intermediatepromoter, a vaccinia early/late promoter and a vaccinia late promoter.

Provided herein is an exemplary vaccinia virus that expresses the humanplasminogen kringle 5 domain under the control of a vaccinia syntheticearly/late promoter is GLV-1h81. Also provided herein are exemplaryvaccinia viruses that express sIL-6R-IL-6 under the control of avaccinia early promoter, vaccinia early/late promoter or vaccinia latepromoter (GLV-1h90, GLV-1h91, and GLV-1h92, respectively). Also providedherein are exemplary vaccinia viruses that express IL-24 under thecontrol of a vaccinia early promoter, vaccinia early/late promoter orvaccinia late promoter (GLV-1h96, GLV-1h97, and GLV-1h98, respectively).Also provided herein are exemplary vaccinia viruses that express atTF-RGD fusion protein under the control of a vaccinia early promoter,vaccinia early/late promoter or vaccinia late promoter (GLV-1h104,GLV-1h105, and GLV-1h106, respectively). Also provided herein areexemplary vaccinia viruses that express an anti-VEGF scAb (G6)-FLAGfusion protein under the control of a vaccinia early promoter, vacciniaearly/late promoter or vaccinia late promoter (GLV-1h107, GLV-1h108 andGLV-1h109, respectively).

Viruses provided herein for therapeutic and diagnostic use includerecombinant vaccinia viruses that contain a heterologous nucleic acidmolecule that encodes a detectable protein or a protein capable ofinducing a detectable signal. Exemplary of such proteins areluciferases, such as a click beetle luciferase, a Renilla luciferase, ora firefly luciferase, fluorescent proteins, such as a GFP or RFP, orproteins that can bind a contrasting agent, chromophore, or a compoundor ligand that can be detected, such as a transferrin receptor or aferritin. Provided herein are recombinant Lister strain vaccinia virusesthat express click beetle luciferase (CBG99) and RFP (e.g., GLV-1h84).

Provided herein are viruses for therapeutic and diagnostic use thatcontain a heterologous nucleic acid molecule that encodes two or morediagnostic or therapeutic gene products, where the gene products arelinked by a picornavirus 2A element. In one example provided herein, therecombinant vaccinia virus contains a heterologous nucleic acid moleculethat encodes CBG99 is linked by a picornavirus 2A element to a secondheterologous nucleic acid molecule that encodes RFP (e.g., GLV-1h84).

Provided herein are recombinant vaccinia viruses for therapeutic anddiagnostic use that contain a replacement of the A34R gene with the A34Rgene from another vaccinia virus strain. Provided herein is a Listerstrain vaccinia virus, where the A34R gene is replaced by the A34R genefrom vaccinia IHD-J strain (e.g., GLV-1i69). Such replacement increasesthe extracellular enveloped virus (EEV) form of vaccinia virus andincreases the resistance of the virus to neutralizing antibodies.

Provided herein are recombinant vaccinia viruses for therapeutic anddiagnostic use that contain deletion of the A35R gene (e.g., GLV-1j87,GLV-1j88 GLV-1j89).

Provided herein are recombinant vaccinia viruses for therapeutic anddiagnostic use that can be further modified by addition of one or moreadditional heterologous nucleic acid molecules that encode a therapeuticprotein, a detectable protein or a protein capable of inducing adetectable signal. Exemplary of such proteins are luciferases, such as aclick beetle luciferase, a Renilla luciferase, or a firefly luciferase,fluorescent proteins, such as a GFP or RFP, or proteins that can bind acontrasting agent, chromophore, or a compound or ligand that can bedetected, such as a transferrin receptor or a ferritin. Also included insuch methods are insertion heterologous nucleic acid molecules thatencode a therapeutic gene product, such as a cytokine, a chemokine, animmunomodulatory molecule, a single chain antibody, antisense RNA,siRNA, prodrug converting enzyme, a toxin, an antitumor oligopeptide, ananti-cancer polypeptide antibiotic, angiogenesis inhibitor, or tissuefactor. Exemplary antigens include tumor specific antigens,tumor-associated antigens, tissue-specific antigens, bacterial antigens,viral antigens, yeast antigens, fungal antigens, protozoan antigens,parasite antigens and mitogens. The one or more additional heterologousnucleic acid molecules that encode a therapeutic protein, a detectableprotein or a protein capable of inducing a detectable signal can beoperatively linked to a promoter, such a vaccinia virus promoter.

Provided herein are host cells that contains a recombinant virusprovided herein. An exemplary host cell is a tumor cell that contains arecombinant virus provided herein.

Provided herein are pharmaceutical compositions that contain arecombinant virus provided herein and a pharmaceutically acceptable. Thecompositions contain an amount or concentration of the virus suitablefor the intended use, such as therapy, diagnostics or both, and route ofadministration. Provided herein are pharmaceutical compositionsformulated for local or systemic administration. Provided herein arepharmaceutical compositions that contain two or more viruses. Providedherein are pharmaceutical compositions that are formulated foradministration as a vaccine, such a smallpox vaccine.

Provided herein are methods of detecting one or more viruses in asubject involving the steps of: a) administering a pharmaceuticalcomposition provided herein to a subject, where the pharmaceuticalcomposition contains a virus provided herein that expresses a detectableprotein or a protein capable of inducing a detectable signal, and b)detecting the detectable protein or a protein capable of inducing adetectable signal, whereby detection indicates the presence of the virusin the subject. Further, provided herein are methods of detecting atumor in a subject involving the steps of: a) administering apharmaceutical composition provided herein to a subject, where thepharmaceutical composition contains a virus provided herein thatexpresses a detectable protein or a protein capable of inducing adetectable signal, and b) detecting the detectable protein or a proteincapable of inducing a detectable signal, whereby detection indicates thepresence of a tumor in the subject. Methods provided herein fordetection include, but are not limited to, fluorescence imaging,magnetic resonance imaging (MRI), single-photon emission computedtomography (SPECT), positron emission tomography (PET), scintigraphy,gamma camera, a β+ detector, a γ detector, or a combination thereof. Insome examples, two or more two or more detectable proteins or proteinscapable of inducing a detectable signal are detected. For example, twoor more fluorescent or luminescent proteins can be detected sequentiallyor simultaneously at different wavelengths.

Provided herein are methods of treatment of a tumor, cancer ormetastasis by administering a pharmaceutical composition provided hereinto a subject, such as a human subject or an animal subject. For themethods provided herein, administering the pharmaceutical compositioncauses tumor growth to stop or be delayed, causes a reduction in tumorvolume or causes the tumor to be eliminated from the subject.

Exemplary tumors in humans for methods of treatment provided hereininclude, but are not limited to, bladder tumor, breast tumor, prostatetumor, carcinoma, basal cell carcinoma, biliary tract cancer, bladdercancer, bone cancer, brain cancer, CNS cancer, glioma tumor, cervicalcancer, choriocarcinoma, colon and rectum cancer, connective tissuecancer, cancer of the digestive system, endometrial cancer, esophagealcancer, eye cancer, cancer of the head and neck, gastric cancer,intra-epithelial neoplasm, kidney cancer, larynx cancer, leukemia, livercancer, lung cancer, lymphoma, Hodgkin's lymphoma, Non-Hodgkin'slymphoma, melanoma, myeloma, neuroblastoma, oral cavity cancer, ovariancancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectalcancer, renal cancer, cancer of the respiratory system, sarcoma, skincancer, stomach cancer, testicular cancer, thyroid cancer, uterinecancer, and cancer of the urinary system. Exemplary tumors in a canine,feline, or pet subject for methods of treatment provided herein include,but are not limited to, lymphosarcoma, osteosarcoma, mammary tumors,mastocytoma, brain tumor, melanoma, adenosquamous carcinoma, carcinoidlung tumor, bronchial gland tumor, bronchiolar adenocarcinoma, fibroma,myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma,retinoblastoma, Ewing's sarcoma, Wilm's tumor, Burkitt's lymphoma,microglioma, neuroblastoma, osteoclastoma, oral neoplasia, fibrosarcoma,osteosarcoma and rhabdomyosarcoma, genital squamous cell carcinoma,transmissible venereal tumor, testicular tumor, seminoma, Sertoli celltumor, hemangiopericytoma, histiocytoma, chloroma, granulocytic sarcoma,corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma,pleural mesothelioma, basal cell tumor, thymoma, stomach tumor, adrenalgland carcinoma, oral papillomatosis, hemangioendothelioma, cystadenoma,follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma, andpulmonary squamous cell carcinoma. Exemplary tumors in a rodent subjectfor methods of treatment provided herein include, but are not limitedto, insulinoma, lymphoma, sarcoma, neuroma, pancreatic islet cell tumor,gastric MALT lymphoma and gastric adenocarcinoma. Exemplary tumors in anovine, equine, bovine, caprine, avian, porcine, or piscine subject formethods of treatment provided herein include, but are not limited to,leukemia, hemangiopericytoma, ocular neoplasia, preputial fibrosarcoma,ulcerative squamous cell carcinoma, preputial carcinoma, connectivetissue neoplasia, mastocytoma, hepatocellular carcinoma, lymphoma,pulmonary adenomatosis, pulmonary sarcoma, Rous sarcoma,reticulo-endotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphoma,lymphoid leukosis, retinoblastoma, hepatic neoplasia, lymphosarcoma,plasmacytoid leukemia, swimbladder sarcoma (in fish), caseouslumphadenitis, and lung tumor.

For the methods provided herein for a therapeutic or diagnosticapplication, a pharmaceutical composition provided herein can beadministered systemically, intravenously, intraarterially,intratumorally, endoscopically, intralesionally, intramuscularly,intradermally, intraperitoneally, intravesicularly, intraarticularly,intrapleurally, percutaneously, subcutaneously, orally, parenterally,intranasally, intratracheally, by inhalation, intracranially,intraprostaticaly, intravitreally, topically, ocularly, vaginally, orrectally.

For the methods provided herein for treatment of a tumor, cancer ormetastasis, the pharmaceutical composition provided herein can beadministered with an anti-viral agent, such as, but not limited to,cidofovir, alkoxyalkyl esters of cidofovir, Gleevec, gancyclovir,acyclovir and ST-26.

Provided herein are combinations that contain a pharmaceuticalcomposition provided herein and an anticancer agent. Exemplaryanticancer agents for use in combinations provided herein include, butare not limited to, a cytokine, a chemokine, a growth factor, aphotosensitizing agent, a toxin, an anti-cancer antibiotic, achemotherapeutic compound, a radionuclide, an angiogenesis inhibitor, asignaling modulator, an anti-metabolite, an anti-cancer vaccine, ananti-cancer oligopeptide, a mitosis inhibitor protein, an antimitoticoligopeptide, an anti-cancer antibody, an anti-cancer antibiotic, animmunotherapeutic agent, hyperthermia or hyperthermia therapy, abacterium, radiation therapy or a combination thereof. Exemplarychemotherapeutic compounds for use in combinations provided hereininclude, but are not limited to, alkylating agents such as a platinumcoordination complex, among other chemotherapeutic compounds providedherein. Exemplary platinum coordination complexes include, but are notlimited to, cisplatin, carboplatin, oxaliplatin, DWA2114R, NK121, IS 3295, and 254-S.

Provided herein are combinations of the viruses provided and an anticancer agent, such as a cytokine, a chemokine, a growth factor, aphotosensitizing agent, a toxin, an anti-cancer antibiotic, achemotherapeutic compound, a radionuclide, an angiogenesis inhibitor, asignaling modulator, an anti-metabolite, an anti-cancer vaccine, ananti-cancer oligopeptide, a mitosis inhibitor protein, an antimitoticoligopeptide, an anti-cancer antibody, an anti-cancer antibiotic, animmunotherapeutic agent, hyperthermia or hyperthermia therapy or abacterium. Provided herein are combinations of the viruses provided andan anti cancer agent, such as cisplatin, carboplatin, gemcitabine,irinotecan, an anti-EGFR antibody and an anti-VEGF antibody.

Provided herein are combinations where the compound and virus areformulated separately in two compositions. Provided herein arecombinations where the compound and virus are formulated as a singlecomposition.

Provided herein are uses of the viruses provided herein for thetreatment of a tumor, cancer or metastasis. Also provided herein areuses of the viruses provided herein for preparation of a pharmaceuticalcomposition for the treatment of a tumor, cancer or metastasis.

Provided herein are kits that contain a pharmaceutical composition orcombination provided herein and optionally instructions foradministration thereof for treatment of cancer.

Provided herein are vaccines, such as a smallpox vaccine, containing arecombinant vaccinia virus provided herein. Further, provided herein aremethods of vaccination where a vaccine, such as a smallpox vaccine,containing a recombinant vaccinia virus provided herein is administeredto a subject for generation of an immune response.

DETAILED DESCRIPTION Outline A. Definitions B. Viruses for treatment anddiagnosis 1. Viruses with altered infectivity a. Viruses with modifiedviral proteins i. Increase in the Vaccinia EEV form by replacement ofA34R ii. Deletion of A35R b. Viruses with multiple genome insertionsand/or deletions 2. Viruses that express proteins for tumor imaging 3.Viruses that express proteins for tumor treatment a. Proteins forinhibiting angiogenesis i. hk5 ii. tTF-RGD iii. anti-VEGF scab b.Proteins for tumor growth suppression i. sIL-6R-IL-6 ii. IL-24 4.Viruses that express proteins for combined tumor diagnosis and treatmentC. Methods of modulating virus attenuation 1. Expression cassettes formodulation of attenuation a. Characteristics of an expression cassettei. Expression cassette promoters ii. Insertion sites for expressioncassettes b. Insertion and/or removal of expression cassettes c.Modification of expression cassettes i. Promoter modification ii.Modification of open reading frame 2. Transcription factor decoys 3.Fine tuning attenuation - Combinations of insertions, deletions and/ormodifications 4. Assays for attenuated viruses D. Further modificationsof viruses provided 1. Modification of viral genes 2. Expression ofadditional heterologous genes a. Detectable gene product b. Therapeuticgene product c. Superantigen d. Gene product to be harvested e. Controlof heterologous gene expression E. Methods for making a modifiedvirus 1. Genetic modifications 2. Screening of modified viruses F.Viruses for use in the methods 1. Cytoplasmic Viruses a. Poxviruses i.Vaccinia Virus b. Other cytoplasmic viruses 2. Adenovirus, Herpes,Retroviruses G. Exemplary characteristics of the viruses provided 1.Attenuated a. Reduced toxicity b. Accumulate in tumor, not substantiallyin other organs c. Ability to elicit or enhance immune response to tumorcells d. Balance of pathogenicity and release of tumor antigens 2.Immunogenicity 3. Replication competent 4. Genetic variants H.Pharmaceutical Compositions, combinations and kits 1. Pharmaceuticalcompositions 2. Host cells 3. Combinations 4. Kits I. TherapeuticMethods 1. Administration a. Steps prior to administering the virus b.Mode of administration c. Dosages d. Number of administrations e.Co-administrations i. Administering a plurality of viruses ii.Therapeutic Compounds iii. Immunotherapies and biological therapies f.State of subject 2. Monitoring a. Monitoring viral gene expression b.Monitoring tumor size c. Monitoring antibody titer d. Monitoring generalhealth diagnostics e. Monitoring coordinated with treatment J. Methodsof producing gene products and antibodies 1. Production of recombinantproteins and RNA molecules 2. Production of antibodies K. Examples

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, websites and other publishedmaterials referred to throughout the entire disclosure herein, unlessnoted otherwise, are incorporated by reference in their entirety. In theevent that there are pluralities of definitions for terms herein, thosein this section prevail. Where reference is made to a URL or other suchidentifier or address, it is understood that such identifiers can changeand particular information on the internet can come and go, butequivalent information is known and can be readily accessed, such as bysearching the internet and/or appropriate databases. Reference theretoevidences the availability and public dissemination of such information.

As used herein, “virus” refers to any of a large group of entitiesreferred to as viruses. Viruses typically contain a protein coatsurrounding an RNA or DNA core of genetic material, but no semipermeablemembrane, and are capable of growth and multiplication only in livingcells. Viruses for use in the methods provided herein include, but arenot limited, to a poxvirus, adenovirus, herpes simplex virus, Newcastledisease virus, vesicular stomatitis virus, mumps virus, influenza virus,measles virus, reovirus, human immunodeficiency virus (HIV), hantavirus, myxoma virus, cytomegalovirus (CMV), lentivirus, and any plant orinsect virus.

As used herein, the term “viral vector” is used according to itsart-recognized meaning. It refers to a nucleic acid vector constructthat includes at least one element of viral origin and can be packagedinto a viral vector particle. The viral vector particles can be used forthe purpose of transferring DNA, RNA or other nucleic acids into cellseither in vitro or in vivo. Viral vectors include, but are not limitedto, retroviral vectors, vaccinia vectors, lentiviral vectors, herpesvirus vectors (e.g., HSV), baculoviral vectors, cytomegalovirus (CMV)vectors, papillomavirus vectors, simian virus (SV40) vectors, semlikiforest virus vectors, phage vectors, adenoviral vectors, andadeno-associated viral (AAV) vectors.

As used herein, the term “modified” with reference to a gene refers to adeleted gene, a gene encoding a gene product having one or moretruncations, mutations, insertions or deletions, or a gene that isinserted (into the chromosome or on a plasmid, phagemid, cosmid, andphage) encoding a gene product, typically accompanied by at least achange in function of the modified gene product or virus.

As used herein, the term “modified virus” refers to a virus that isaltered with respect to a parental strain of the virus. Typicallymodified viruses have one or more truncations, mutations, insertions ordeletions in the genome of virus. A modified virus can have one or moreendogenous viral genes modified and/or one or more intergenic regionsmodified. Exemplary modified viruses can have one or more heterologousnucleic acid sequences inserted into the genome of the virus. Modifiedviruses can contain one more heterologous nucleic acid sequences in theform of a gene expression cassette for the expression of a heterologousgene.

As used herein, modification of a heterologous nucleic acid moleculewith respect to a virus containing a heterologous nucleic acid moleculerefers to any alteration of the heterologous nucleic acid moleculeincluding truncations, mutations, insertions or deletions of the nucleicacid molecule. A deletion in a heterologous nucleic acid molecule caninclude all or a portion of the heterologous nucleic acid molecule. Forexample, if the heterologous nucleic acid molecule is a double strandedDNA molecule that is 5,000 base pairs in length, deletions of theheterologous nucleic acid molecule can include deletions of 1, 2, 3, 4,5 or more, 10 or more, 50 or more, 100 or more, 500 or more, 1,000 ormore, or 5,000 base pairs of the heterologous nucleic acid molecule.Deletion of all or a part of the nucleic acid molecule can also includereplacement of the heterologous nucleic acid molecule with anothernucleic acid molecule. Modification of a heterologous nucleic acidmolecule can also include alteration of the viral genome. For example, adeletion of all or a portion heterologous nucleic from the viral genome,for example by homologous recombination, may also include deletion ofnucleic acid surrounding the deletion site that is part of the viralgenome. Similarly, insertion of an additional heterologous nucleic acidmolecule into the viral genome by homologous recombination, for example,may include deletion or all, or a part of a viral gene. Whenmodification of a heterologous nucleic acid molecule is an insertion, anadditional nucleic acid molecule can be inserted in the heterologousnucleic acid molecule or adjacent to the nucleic acid molecule.Typically, insertions by homologous recombination involve replacement ofall or a part of the heterologous nucleic acid molecule with anothernucleic acid molecule.

As used herein, the term “therapeutic virus” refers to a virus that isadministered for the treatment of a disease or disorder, such as cancer,a tumor and/or a metastasis or inflammation or wound or diagnosisthereof and or both. The A therapeutic virus typically is modified, suchas to attenuate it. Other modifications include one or more insertions,deletions or mutations in the genome of the virus. Therapeutic virusesall can include modifications in one or more endogenous viral genes orone or more intergenic regions, which attenuate the toxicity of thevirus, and can optionally express a heterologous therapeutic geneproduct and/or detectable protein. Therapeutic viruses can containheterologous nucleic acid molecules, including one or more geneexpression cassettes for the expression of the therapeutic gene productand/or detectable protein. Therapeutic viruses can be replicationcompetent viruses (e.g., oncolytic viruses) or replication-defectiveviruses.

As used herein, a virus that can be detected and used for diagnosticsand is therapeutic is a theragnostic virus.

As used herein, the term, “therapeutic gene product” or “therapeuticpolypeptide” refers to any heterologous protein expressed by thetherapeutic virus that ameliorates the symptoms of a disease or disorderor ameliorates the disease or disorder.

As used herein, the phrase “immunoprivileged cells and tissues” refersto cells and tissues, such as solid tumors and wounded tissues, whichare sequestered from the immune system.

As used herein, preferential accumulation refers to accumulation of avirus at a first location at a higher level than accumulation at asecond location. Thus, a virus that preferentially accumulates inimmunoprivileged tissue, such as a tumor, relative to normal tissues ororgans refers to a virus that accumulates in immunoprivileged tissue,such as tumor, at a higher level, or concentration, than the virusaccumulates in normal tissues or organs.

As used herein, to attenuate toxicity of a virus means to reduce oreliminate deleterious or toxic effects to a host upon administration ofthe virus compared to an un-attenuated virus. As used herein, a viruswith low toxicity means that upon administration a virus does notaccumulate in organs and tissues in the host to an extent that resultsin damage or harm to organs, or that impacts survival of the host to agreater extent than the disease being treated does. For the purposesherein, attenuation of toxicity is used interchangeably with attenuationof virulence and attenuation of pathogenicity.

As used herein, the term “toxicity” with reference to a virus refers tothe ability of the virus to cause harm to the subject to which the virushas been administered.

As used herein virulence and pathogenicity with reference to a virusrefers to the ability of the virus to cause disease or harm in thesubject to which the virus has been administered. Hence, for thepurposes herein the terms toxicity, virulence and pathogenicity withreference to a virus are used interchangeably.

As used herein, a compound produced in a tumor or other immunoprivilegedsite refers to any compound that is produced in the tumor or tumorenvironment by virtue of the presence of an introduced virus, generallya recombinant virus, expressing one or more gene products. For example,a compound produced in a tumor can be, for example, an encodedpolypeptide, such as a recombinant polypeptide (e.g., a cell-surfacereceptor, a cytokine, a chemokine, an apoptotic protein, a mitosisinhibitor protein, an antimitotic oligopeptide, a toxin, a tumorantigen, a prodrug converting enzyme), an RNA (e.g., ribozyme, RNAi,siRNA), or a compound that is generated by an encoded polypeptide and,in some examples, the cellular machinery of the tumor orimmunoprivileged tissue or cells (e.g., a metabolite, a convertedprodrug).

As used herein, a delivery vehicle for administration refers to alipid-based or other polymer-based composition, such as liposome,micelle or reverse micelle, which associates with an agent, such as avirus provided herein, for delivery into a host animal.

As used herein, a disease or disorder refers to a pathological conditionin an organism resulting from, for example, infection or genetic defect,and characterized by identifiable symptoms.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered. Treatment also encompasses any pharmaceutical use of theviruses described and provided herein.

As used herein, amelioration or alleviation of the symptoms of aparticular disorder, such as by administration of a particularpharmaceutical composition, refers to any lessening, whether permanentor temporary, lasting or transient that can be attributed to orassociated with administration of the composition.

As used herein, an effective amount of a virus or compound for treatinga particular disease is an amount that is sufficient to ameliorate, orin some manner reduce the symptoms associated with the disease. Such anamount can be administered as a single dosage or can be administeredaccording to a regimen, whereby it is effective. The amount can cure thedisease but, typically, is administered in order to ameliorate thesymptoms of the disease. Repeated administration can be required toachieve the desired amelioration of symptoms.

As used herein, an in vivo method refers to a method performed withinthe living body of a subject.

As used herein, a subject includes any animal for whom diagnosis,screening, monitoring or treatment is contemplated. Animals includemammals such as primates and domesticated animals. An exemplary primateis human. A patient refers to a subject such as a mammal, primate,human, or livestock subject afflicted with a disease condition or forwhich a disease condition is to be determined or risk of a diseasecondition is to be determined.

As used herein, the term “neoplasm” or “neoplasia” refers to abnormalnew cell growth, and thus means the same as tumor, which can be benignor malignant. Unlike hyperplasia, neoplastic proliferation persists evenin the absence of the original stimulus.

As used herein, neoplastic disease refers to any disorder involvingcancer, including tumor development, growth, metastasis and progression.

As used herein, cancer is a term for diseases caused by or characterizedby any type of malignant tumor, including metastatic cancers, lymphatictumors, and blood cancers. Exemplary cancers include, but are notlimited to: leukemia, lymphoma, pancreatic cancer, lung cancer, ovariancancer, breast cancer, cervical cancer, bladder cancer, prostate cancer,glioma tumors, adenocarcinomas, liver cancer and skin cancer. Exemplarycancers in humans include a bladder tumor, breast tumor, prostate tumor,basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer,brain and CNS cancer (e.g., glioma tumor), cervical cancer,choriocarcinoma, colon and rectum cancer, connective tissue cancer,cancer of the digestive system; endometrial cancer, esophageal cancer;eye cancer; cancer of the head and neck; gastric cancer;intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemia; livercancer; lung cancer (e.g. small cell and non-small cell); lymphomaincluding Hodgkin's and Non-Hodgkin's lymphoma; melanoma; myeloma,neuroblastoma, oral cavity cancer (e.g., lip, tongue, mouth, andpharynx); ovarian cancer; pancreatic cancer, retinoblastoma;rhabdomyosarcoma; rectal cancer, renal cancer, cancer of the respiratorysystem; sarcoma, skin cancer; stomach cancer, testicular cancer, thyroidcancer; uterine cancer, cancer of the urinary system, as well as othercarcinomas and sarcomas. Malignant disorders commonly diagnosed in dogs,cats, and other pets include, but are not limited to, lymphosarcoma,osteosarcoma, mammary tumors, mastocytoma, brain tumor, melanoma,adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor,bronchiolar adenocarcinoma, fibroma, myxochondroma, pulmonary sarcoma,neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma,Wilm's tumor, Burkitt's lymphoma, microglioma, neuroblastoma,osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma andrhabdomyosarcoma, genital squamous cell carcinoma, transmissiblevenereal tumor, testicular tumor, seminoma, Sertoli cell tumor,hemangiopericytoma, histiocytoma, chloroma (e.g., granulocytic sarcoma),corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma,pleural mesothelioma, basal cell tumor, thymoma, stomach tumor, adrenalgland carcinoma, oral papillomatosis, hemangioendothelioma andcystadenoma, follicular lymphoma, intestinal lymphosarcoma, fibrosarcomaand pulmonary squamous cell carcinoma. In rodents, such as a ferret,exemplary cancers include insulinoma, lymphoma, sarcoma, neuroma,pancreatic islet cell tumor, gastric MALT lymphoma and gastricadenocarcinoma. Neoplasias affecting agricultural livestock includeleukemia, hemangiopericytoma and bovine ocular neoplasia (in cattle);preputial fibrosarcoma, ulcerative squamous cell carcinoma, preputialcarcinoma, connective tissue neoplasia and mastocytoma (in horses);hepatocellular carcinoma (in swine); lymphoma and pulmonary adenomatosis(in sheep); pulmonary sarcoma, lymphoma, Rous sarcoma,reticulo-endotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphomaand lymphoid leukosis (in avian species); retinoblastoma, hepaticneoplasia, lymphosarcoma (lymphoblastic lymphoma), plasmacytoid leukemiaand swimbladder sarcoma (in fish), caseous lumphadenitis (CLA): chronic,infectious, contagious disease of sheep and goats caused by thebacterium Corynebacterium pseudotuberculosis, and contagious lung tumorof sheep caused by jaagsiekte.

As used herein, the term “malignant,” as it applies to tumors, refers toprimary tumors that have the capacity of metastasis with loss of growthcontrol and positional control.

As used herein, metastasis refers to a growth of abnormal or neoplasticcells distant from the site primarily involved by the morbid process.

As used herein, proliferative disorders include any disorders involvingabnormal proliferation of cells, such as, but not limited to, neoplasticdiseases.

As used herein, a method for treating or preventing neoplastic diseasemeans that any of the symptoms, such as the tumor, metastasis thereof,the vascularization of the tumors or other parameters by which thedisease is characterized are reduced, ameliorated, prevented, placed ina state of remission, or maintained in a state of remission. It alsomeans that the indications of neoplastic disease and metastasis can beeliminated, reduced or prevented by the treatment. Non-limiting examplesof the indications include uncontrolled degradation of the basementmembrane and proximal extracellular matrix, migration, division, andorganization of the endothelial cells into new functioning capillaries,and the persistence of such functioning capillaries.

As used herein, the term “angiogenesis” is intended to encompass thetotality of processes directly or indirectly involved in theestablishment and maintenance of new vasculature (neovascularization),including, but not limited to, neovascularization associated with tumorsand neovascularization associated with wounds.

As used herein, therapeutic agents are agents that ameliorate thesymptoms of a disease or disorder or ameliorate the disease or disorder.Therapeutic agent, therapeutic compound, therapeutic regimen, orchemotherapeutic include conventional drugs and drug therapies,including vaccines, which are known to those skilled in the art anddescribed elsewhere herein. Therapeutic agents include, but are notlimited to, moieties that inhibit cell growth or promote cell death,that can be activated to inhibit cell growth or promote cell death, orthat activate another agent to inhibit cell growth or promote celldeath. Optionally, the therapeutic agent can exhibit or manifestadditional properties, such as, properties that permit its use as animaging agent, as described elsewhere herein. Therapeutic agents for thecompositions, methods and uses provided herein can be, for example, ananti-cancer agent. Exemplary therapeutic agents include, for example,cytokines, growth factors, photosensitizing agents, radionuclides,toxins, anti-metabolites, signaling modulators, anti-cancer antibiotics,anti-cancer antibodies, angiogenesis inhibitors, radiation therapy,chemotherapeutic compounds or a combination thereof.

As used herein, anti-cancer agents (used interchangeably with“anti-tumor or anti-neoplastic” agent) include any anti-cancertherapies, such as radiation therapy, surgery, hyperthermia orhyperthermia therapy, or anti-cancer compounds useful in the treatmentof cancer. These include any agents, when used alone or in combinationwith other agent, that can alleviate, reduce, ameliorate, prevent, orplace or maintain in a state of remission of clinical symptoms ordiagnostic markers associated with neoplastic disease, tumors andcancer, and can be used in methods, combinations and compositionsprovided herein. Exemplary anti-cancer agents include, but are notlimited to, the viruses provided herein used singly or in combinationand/or in combination with other anti-cancer agents. Exemplaryanti-cancer compounds include a cytokines, chemokines, growth factors, aphotosensitizing agents, toxins, anti-cancer antibiotics,chemotherapeutic compounds, radionuclides, angiogenesis inhibitors,signaling modulators, anti-metabolites, anti-cancer vaccines,anti-cancer oligopeptides, mitosis inhibitor proteins, antimitoticoligopeptides, anti-cancer antibodies, anti-cancer antibiotics,immunotherapeutic agents, bacteria and any combinations thereof.

Exemplary cytokines and growth factors include, but are not limited to,interleukins, such as, for example, interleukin-1, interleukin-2,interleukin-6 and interleukin-12, tumor necrosis factors, such as tumornecrosis factor alpha (TNF-α), interferons such as interferon gamma(IFN-γ), granulocyte macrophage colony stimulating factors (GM-CSF),angiogenins, and tissue factors.

Photosensitizing agents include, but are not limited to, for example,indocyanine green, toluidine blue, aminolevulinic acid, texaphyrins,benzoporphyrins, phenothiazines, phthalocyanines, porphyrins such assodium porfimer, chlorins such as tetra(m-hydroxyphenyl)chlorin ortin(IV) chlorin e6, purpurins such as tin ethyl etiopurpurin,purpurinimides, bacteriochlorins, pheophorbides, pyropheophorbides orcationic dyes.

Radionuclides, which depending upon the radionuclide, amount andapplication can be used for diagnosis and/or for treatment. Theyinclude, but are not limited to, for example, a compound or moleculecontaining ¹¹Carbon, ¹¹Fluorine, ¹³-Carbon, ¹⁵-Nitrogen, ¹⁸Flourine,¹⁹Flourine, ³²Phosphate, ⁶⁰Cobalt, ⁹⁰Yttirum, ⁹⁹Technetium, ¹⁰³Palladium, ¹⁰⁶Ruthenium, ¹¹¹Indium, ¹¹⁷Lutetium, ¹²⁵Iodine, ¹³¹Iodine,¹³⁷Cesium, ¹⁵³Samarium, ¹⁸⁶Rhenium, ⁸⁸Rhenium, 192Iridium, ¹⁹⁸Gold,²¹¹Astatine, ²¹²Bismuth or ²¹³Bismuth.

Toxins include, but are not limited to, chemotherapeutic compounds suchas, but not limited to, 5-fluorouridine, calicheamicin and maytansine.

Anti-metabolites include, but are not limited to, methotrexate,5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, hydroxyurea and20-chlorodeoxyadenosine.

Signaling modulators include, but are not limited to, for example,inhibitors of macrophage inhibitory factor, toll-like receptor agonistsand stat 3 inhibitors.

Anti-cancer antibiotics include, but are not limited to, anthracyclinessuch as doxorubicin hydrochloride (adriamycin), idarubicinhydrochloride, daunorubicin hydrochloride, aclarubicin Hydrochloride,epirubicin hydrochloride and purarubicin hydrochloride, enomycin,phenomycin, pleomycins such as pleomycin and peplomycin sulfate,mitomycins such as mitomycin C, actinomycins such as actinomycin D,zinostatinstimalamer and polypeptides such as neocarzinostatin.

Anti-cancer antibodies include, but are not limited to, Rituximab,ADEPT, Trastuzumab (Herceptin), Tositumomab (Bexxar), Cetuximab(Erbitux), Ibritumomab (Zevalin), Alemtuzumab (Campath-1H), Epratuzumab(Lymphocide), Gemtuzumab ozogamicin (Mylotarg), Bevacimab (Avastin),Tarceva (Erlotinib), SUTENT (sunitinib malate), Panorex (Edrecolomab),RITUXAN (Rituximab), Zevalin (90Y-ibritumomab tiuexetan), Mylotarg(Gemtuzumab Ozogamicin) and Campath (Alemtuzumab).

Angiogenesis inhibitors include, but are not limited to, collagenaseinhibitors such as metalloproteinases and tetracyclines such asminocycline, naturally occurring peptides such as endostatin andangiostatin, fungal and bacterial derivatives, such as fumagillinderivatives like TNP-470, aptamer antogonist of VEGF, batimastat,Captopril, cartilage derived inhibitor (CDI), genistein, interleukin 12,Lavendustin A, medroxypregesterone acetate, recombinant human plateletfactor 4(rPF4), taxol, D-gluco-D-galactan sulfate (Tecogalan(=SP-PG,DS-4152)), thalidomide, thrombospondin.

Radiation therapy includes, but is not limited to, photodynamic therapy,radionuclides, radioimmunotherapy and proton beam treatment.

Chemotherapeutic compounds include, but are not limited to platinum;platinum analogs (e.g., platinum coordination complexes) such ascisplatin, carboplatin, oxaliplatin, DWA2114R, NK121, IS 3 295, and254-S; anthracenediones; vinblastine; alkylating agents such as thiotepaand cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfanand piposulfan; aziridines such as benzodopa, carboquone, meturedopa anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime nitrogen mustardssuch as chiorambucil, chlomaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; etoglucid; galliumnitrate; substituted ureas; hydroxyurea; lentinan; lonidamine;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine;anti-cancer polysaccharides; polysaccharide-K; razoxane; sizofuran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytosinearabinoside; cyclophosphamide; thiotepa; taxoids, such as paclitaxel anddoxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;methotrexate; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid; esperamicins;capecitabine; methylhydrazine derivatives; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. Alsoincluded in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogensincluding for example tamoxifen, raloxifene, aromatase inhibiting4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,onapristone and toremifene (Fareston); adrenocortical suppressants; andantiandrogens such as flutamide, nilutamide, bicalutamide, leuprolideand goserelin; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Such chemotherapeutic compounds thatcan be used herein include compounds whose toxicities preclude use ofthe compound in general systemic chemotherapeutic methods.

As used herein, an anti-cancer oligopeptide or an anti-tumoroligopeptide is short polypeptide that has the ability to slow orinhibit tumor growth and/or metastasis. Anti-cancer oligopeptidetypically have high affinity for and specificity to tumors enabling themto target tumors. Such oligopeptides include receptor-interactingcompounds, inhibitors of protein-protein interactions, enzymeinhibitors, and nucleic acid-interacting compounds. As used herein anantimitotic oligopeptide is an oligopeptide that inhibits cell division.An antimitotic oligopeptide is an exemplary anti-cancer oligopeptide.Exemplary antimitotic oligopeptides include, but are not limited to,tubulysin, phomopsin, hemiasterlin, taltobulin (HTI-286, 3), andcryptophycin.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound is regenerated by metabolicprocesses. The prodrug can be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound (see, e.g., Nogrady (1985) Medicinal ChemistryA Biochemical Approach, Oxford University Press, New York, pages388-392). Prodrugs include, but are not limited to, 5-fluorocytosine,gancyclovir, 6-methylpurine deoxyriboside, cephalosporin-doxorubicin,4-[(2-chloroethyl)(2-mesuloxyethyl)amino]benzoyl-L-glutamic acid,indole-3-acetic acid,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycampotothecin,bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28,1-chloromethyl-5-hydroxy-1,2-dihyro-3H-benz[e]indole,epirubicin-glucoronide, 5′-deoxy-5-fluorouridine, cytosine arabinoside,and linamarin.

As used herein, a compound conjugated to a moiety refers to a complexthat includes a compound bound to a moiety, where the binding betweenthe compound and the moiety can arise from one or more covalent bonds ornon-covalent interactions such as hydrogen bonds, or electrostaticinteractions. A conjugate also can include a linker that connects thecompound to the moiety. Exemplary compounds include, but are not limitedto, nanoparticles and siderophores. Exemplary moieties, include, but arenot limited to, detectable moieties and therapeutic agents.

As used herein, nanoparticle refers to a microscopic particle whose sizeis measured in nanometers. Often such particles in nanoscale are used inbiomedical applications acting as drug carriers or imaging agents.Nanoparticles can be conjugated to other agents, including, but notlimited to detectable/diagnostic agents or therapeutic agents.

As used herein, a detectable label or detectable moiety or diagnosticmoiety (also imaging label, imaging agent, or imaging moiety) refers toan atom, molecule or composition, wherein the presence of the atom,molecule or composition can be directly or indirectly measured.

As used herein, a detectable moiety or an imaging moiety refer tomoieties used to image a virus in any of the methods provided herein.Imaging (detectable) moieties include, for example, chemiluminescentmoieties, bioluminescent moieties, fluorescent moieties, radionuclidesand metals.

As used herein, a detection agent or an imaging agent refer to anymolecule, compound, or polypeptide used to image a virus in any of themethods provided herein. Detection agents or imaging agents can contain,for example, a detectable moiety or can be a substrate, such as aluciferin, that produces a detectable signal following modification,such as by chemical modification by a luciferase.

As used herein, detect, detected and detecting refer generally to anymanner of discovering or determining the presence of a signal, such asvisual inspection, fluorescence spectroscopy, absorption, reflectancemeasurement, flow cytometry, magnetic resonance methods such as magneticresonance imaging (MRI) and magnetic resonance spectroscopy (MRS),ultrasound, X-rays, gamma rays (after annihilation of a positron and anelectron in PET scanning), tomographic methods including computedtomography (CT), computed axial tomography (CAT), electron beam computedtomography (EBCT), high resolution computed tomography (HRCT),hypocycloidal tomography, positron emission tomography (PET),single-photon emission computed tomography (SPECT), spiral computedtomography and ultrasonic tomography. Direct detection of a detectablelabel refers to, for example, measurement of a physical phenomenon, suchas energy or particle emission or absorption of the moiety itself, suchas by X-ray or MRI. Indirect detection refers to measurement of aphysical phenomenon, such as energy or particle emission or absorption,of an atom, molecule or composition that binds directly or indirectly tothe detectable moiety. In a non-limiting example of indirect detection,a detectable label can be biotin, which can be detected by binding toavidin. Non-labeled avidin can be administered systemically to blocknon-specific binding, followed by systemic administration of labeledavidin. Thus, included within the scope of a detectable label ordetectable moiety is a bindable label or bindable moiety, which refersto an atom, molecule or composition, wherein the presence of the atom,molecule or composition can be detected as a result of the label ormoiety binding to another atom, molecule or composition. Exemplarydiagnostic agents include, for example, metals such as colloidal gold,iron, gadolinium, and gallium-67, fluorescent moieties andradionuclides. Exemplary fluorescent moieties and radionuclides areprovided elsewhere herein.

As used herein, magnetic resonance imaging (MRI) refers to the use of anuclear magnetic resonance spectrometer to produce electronic images ofspecific atoms and molecular structures in solids, especially humancells, tissues and organs. MRI is non-invasive diagnostic technique thatuses nuclear magnetic resonance to produce cross-sectional images oforgans and other internal body structures. The subject lies inside alarge, hollow cylinder containing a strong electromagnet, which causesthe nuclei of certain atoms in the body (such as, for example, ¹H, ¹³Cand 19F) to align magnetically. The subject is then subjected to radiowaves, which cause the aligned nuclei to flip; when the radio waves arewithdrawn the nuclei return to their original positions, emitting radiowaves that are then detected by a receiver and translated into atwo-dimensional picture by computer. For some MRI procedures, contrastagents such as gadolinium are used to increase the accuracy of theimages.

As used herein, an X-ray refers to a relatively high-energy photon, or astream of such photons, having a wavelength in the approximate rangefrom 0.01 to 10 nanometers. X-rays also refer to photographs taken withx-rays.

As used herein, nucleic acids include DNA, RNA and analogs thereof,including peptide nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double-stranded. Nucleic acids can encode forexample gene products, such as, for example, polypeptides, regulatoryRNAs, siRNAs and functional RNAs.

As used herein, primer refers to an oligonucleotide containing two ormore deoxyribonucleotides or ribonucleotides, typically more than three,from which synthesis of a primer extension product can be initiated.Typically a primer contains a free 3′ hydroxy moiety. Experimentalconditions conducive to synthesis of a gene product include the presenceof nucleoside triphosphates and an agent for polymerization andextension, such as DNA polymerase, and a suitable buffer, temperature,and pH. When referring to probes or primers, which are optionallylabeled, such as with a detectable label, such as a fluorescent orradiolabel, single-stranded molecules are provided. Such molecules aretypically of a length such that their target is statistically unique orof low copy number (typically less than 5, generally less than 3) forprobing or priming a library. Generally a probe or primer contains atleast 14, 16 or 30 contiguous nucleotides of sequence complementary toor identical to a gene of interest. Probes and primers can be 5, 6, 7,8, 9, 10 or more, 20 or more, 30 or more, 50 or more, 100 or morenucleic acids long.

As used herein, a sequence complementary to at least a portion of anRNA, with reference to antisense oligonucleotides, means a sequence ofnucleotides having sufficient complementarity to be able to hybridizewith the RNA, generally under moderate or high stringency conditions,forming a stable duplex; in the case of double-stranded antisensenucleic acids, a single strand of the duplex DNA (i.e., dsRNA) can thusbe tested, or triplex formation can be assayed. The ability to hybridizedepends on the degree of complementarily and the length of the antisensenucleic acid. Generally, the longer the hybridizing nucleic acid, themore base mismatches with an encoding RNA it can contain and still forma stable duplex (or triplex, as the case can be). One skilled in the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

As used herein, a heterologous nucleic acid (also referred to asexogenous nucleic acid or foreign nucleic acid) refers to a nucleic acidthat is not normally produced in vivo by an organism or virus from whichit is expressed or that is produced by an organism or a virus but is ata different locus, expressed differently, or that mediates or encodesmediators that alter expression of endogenous nucleic acid, such as DNA,by affecting transcription, translation, or other regulatablebiochemical processes. Heterologous nucleic acid is often not endogenousto a cell or virus into which it is introduced, but has been obtainedfrom another cell or virus or prepared synthetically. Heterologousnucleic acid can refer to a nucleic acid molecule from another cell inthe same organism or another organism, including the same species oranother species. Heterologous nucleic acid, however, can be endogenous,but is nucleic acid that is expressed from a different locus or alteredin its expression or sequence (e.g., a plasmid). Thus, heterologousnucleic acid includes a nucleic acid molecule not present in the exactorientation or position as the counterpart nucleic acid molecule, suchas DNA, is found in a genome. Generally, although not necessarily, suchnucleic acid encodes RNA and proteins that are not normally produced bythe cell or virus or in the same way in the cell in which it isexpressed. Any nucleic acid, such as DNA, that one of skill in the artrecognizes or considers as heterologous, exogenous or foreign to thecell in which the nucleic acid is expressed is herein encompassed byheterologous nucleic acid.

As used herein, a heterologous protein or heterologous polypeptide (alsoreferred to as exogenous protein, exogenous polypeptide, foreign proteinor foreign polypeptide) refers to a protein that is not normallyproduced in vivo by an organism.

As used herein, operative linkage of heterologous nucleic acids toregulatory and effector sequences of nucleotides, such as promoters,enhancers, transcriptional and translational stop sites, and othersignal sequences refers to the relationship between such nucleic acid,such as DNA, and such sequences of nucleotides. For example, operativelinkage of heterologous DNA to a promoter refers to the physicalrelationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. Thus, operatively linked or operationally associated refers to thefunctional relationship of a nucleic acid, such as DNA, with regulatoryand effector sequences of nucleotides, such as promoters, enhancers,transcriptional and translational stop sites, and other signalsequences. For example, operative linkage of DNA to a promoter refers tothe physical and functional relationship between the DNA and thepromoter such that the transcription of such DNA is initiated from thepromoter by an RNA polymerase that specifically recognizes, binds to andtranscribes the DNA. In order to optimize expression and/ortranscription, it can be necessary to remove, add or alter 5′untranslated portions of the clones to eliminate extra, potentiallyinappropriate, alternative translation initiation (i.e., start) codonsor other sequences that can interfere with or reduce expression, eitherat the level of transcription or translation. In addition, consensusribosome binding sites can be inserted immediately 5′ of the start codonand can enhance expression (see, e.g., Kozak J. Biol. Chem. 266:19867-19870 (1991); Shine and Delgarno Nature 254(5495): 34-38 (1975)).The desirability of (or need for) such modification can be empiricallydetermined.

As used herein, a promoter, a promoter region or a promoter element orregulatory region or regulatory element refers to a segment of DNA orRNA that controls transcription of the DNA or RNA to which it isoperatively linked. The promoter region includes specific sequences thatare involved in RNA polymerase recognition, binding and transcriptioninitiation. In addition, the promoter includes sequences that modulaterecognition, binding and transcription initiation activity of RNApolymerase (i.e., binding of one or more transcription factors). Thesesequences can be cis acting or can be responsive to trans actingfactors. Promoters, depending upon the nature of the regulation, can beconstitutive or regulated. Regulated promoters can be inducible orenvironmentally responsive (e.g. respond to cues such as pH, anaerobicconditions, osmoticum, temperature, light, or cell density). Many suchpromoter sequences are known in the art. See, for example, U.S. Pat.Nos. 4,980,285; 5,631,150; 5,707,928; 5,759,828; 5,888,783; 5,919,670,and, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Press (1989).

As used herein, a native promoter is a promoter that is endogenous tothe organism or virus and is unmodified with respect to its nucleotidesequence and its position in the viral genome as compared to a wild-typeorganism or virus.

As used herein, a heterologous promoter refers to a promoter that is notnormally found in the wild-type organism or virus or that is at adifferent locus as compared to a wild-type organism or virus. Aheterologous promoter is often not endogenous to a cell or virus intowhich it is introduced, but has been obtained from another cell or virusor prepared synthetically. A heterologous promoter can refer to apromoter from another cell in the same organism or another organism,including the same species or another species. A heterologous promoter,however, can be endogenous, but is a promoter that is altered in itssequence or occurs at a different locus (e.g., at a different locationin the genome or on a plasmid). Thus, a heterologous promoter includes apromoter not present in the exact orientation or position as thecounterpart promoter is found in a genome.

A synthetic promoter is a heterologous promoter that has a nucleotidesequence that is not found in nature. A synthetic promoter can be anucleic acid molecule that has a synthetic sequence or a sequencederived from a native promoter or portion thereof. A synthetic promotercan also be a hybrid promoter composed of different elements derivedfrom different native promoters.

As used herein a “gene expression cassette” or “expression cassette” isa nucleic acid construct, containing nucleic acid elements that arecapable of effecting expression of a gene in hosts that are compatiblewith such sequences. Expression cassettes include at least promoters andoptionally, transcription termination signals. Typically, the expressioncassette includes a nucleic acid to be transcribed operably linked to apromoter. Additional factors helpful in effecting expression can also beused as described herein. Expression cassettes can contain genes thatencode, for example, a therapeutic gene product or a detectable proteinor a selectable marker gene,

As used herein, replacement of a promoter with a stronger promoterrefers to removing a promoter from a genome and replacing it with apromoter that effects an increased the level of transcription initiationrelative to the promoter that is replaced. Typically, a strongerpromoter has an improved ability to bind polymerase complexes relativeto the promoter that is replaced. As a result, an open reading framethat is operably linked to the stronger promoter has a higher level ofgene expression. Similarly, replacement of a promoter with a weakerpromoter refers to removing a promoter from a genome and replacing itwith a promoter that decreases the level of transcription initiationrelative to the promoter that is replaced. Typically, a weaker promoterhas a lessened ability to bind polymerase complexes relative to thepromoter that is replaced. As a result, an open reading frame that isoperably linked to the weaker promoter has a lower level of geneexpression.

As used herein, production by recombinant means by using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous nucleic acid into cells for eitherexpression or replication thereof. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Selection and use of suchvectors are well known to those of skill in the art. An expressionvector includes vectors capable of expressing DNA that is operativelylinked with regulatory sequences, such as promoter regions, that arecapable of effecting expression of such DNA fragments. Thus, anexpression vector refers to a recombinant DNA or RNA construct, such asa plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome. Vectors can be used in thegeneration of a recombinant genome by integration or homologousrecombination, such as in the generation of a recombinant virus asdescribed elsewhere herein.

As used herein, genetic therapy or gene therapy involves the transfer ofheterologous nucleic acid, such as DNA or RNA, into certain cells,target cells, of a mammal, particularly a human, with a disorder orconditions for which such therapy is sought. As used herein, genetictherapy or gene therapy can involve the transfer of heterologous nucleicacid, such as DNA, into a virus, which can be transferred to a mammal,particularly a human, with a disorder or conditions for which suchtherapy is sought. The nucleic acid, such as DNA, is introduced into theselected target cells, such as directly or indirectly, in a manner suchthat the heterologous nucleic acid, such as DNA, is expressed and atherapeutic product encoded thereby is produced. Alternatively, theheterologous nucleic acid, such as DNA, can in some manner mediateexpression of DNA that encodes the therapeutic product, or it can encodea product, such as a peptide or RNA that is in some manner a therapeuticproduct, or which mediates, directly or indirectly, expression of atherapeutic product. Genetic therapy also can be used to deliver nucleicacid encoding a gene product that replaces a defective gene orsupplements a gene product produced by the mammal or the cell in whichit is introduced. The introduced nucleic acid can encode a therapeuticcompound. The heterologous nucleic acid, such as DNA, encoding thetherapeutic product can be modified prior to introduction into the cellsof the afflicted host in order to enhance or otherwise alter the productor expression thereof. Genetic therapy also can involve delivery of aninhibitor or repressor or other modulator of gene expression.

As used herein, a therapeutically effective product for gene therapy isa product that is encoded by heterologous nucleic acid, typically DNA,or an RNA product such as dsRNA, RNAi, including siRNA, that uponintroduction of the nucleic acid into a host, a product is expressedthat ameliorates or eliminates the symptoms, manifestations of aninherited or acquired disease or that cures the disease. Also includedare biologically active nucleic acid molecules, such as RNAi andantisense nucleic acids.

As used herein, an agent or compound that modulates the activity of aprotein or expression of a gene or nucleic acid either decreases orincreases or otherwise alters the activity of the protein or, in somemanner, up- or down-regulates or otherwise alters expression of thenucleic acid in a cell.

As used herein, recitation that amino acids of a polypeptide “correspondto” amino acids in a disclosed sequence, such as amino acids set forthin the Sequence listing, refers to amino acids identified upon alignmentof the polypeptide with the disclosed sequence to maximize identity orhomology (where conserved amino acids are aligned) using a standardalignment algorithm, such as the GAP algorithm. By aligning thesequences of polypeptides, one skilled in the art can identifycorresponding residues, using conserved and identical amino acidresidues as guides.

As used herein, “amino acids” are represented by their full name or bytheir known, three-letter or one-letter abbreviations (Table 1). Thenucleotides which occur in the various nucleic acid fragments aredesignated with the standard single-letter designations used routinelyin the art.

TABLE 1 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr tyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine XXaa Unknown or other

As used herein, the terms “homology” and “identity” are usedinterchangeably, but homology for proteins can include conservativeamino acid changes. In general, to identify corresponding positions, thesequences of amino acids are aligned so that the highest order match isobtained (see, e.g., Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; Carillo et al. (1988) SIAM J Applied Math 48:1073).

As use herein, “sequence identity” refers to the number of identicalamino acids (or nucleotide bases) in a comparison between a test and areference polypeptide or polynucleotide. Homologous polypeptides referto a pre-determined number of identical or homologous amino acidresidues. Homology includes conservative amino acid substitutions aswell identical residues. Sequence identity can be determined by standardalignment algorithm programs used with default gap penalties establishedby each supplier. Homologous nucleic acid molecules refer to apre-determined number of identical or homologous nucleotides. Homologyincludes substitutions that do not change the encoded amino acid (i.e.,“silent substitutions”) as well identical residues. Substantiallyhomologous nucleic acid molecules hybridize typically at moderatestringency or at high stringency all along the length of the nucleicacid or along at least about 70%, 80% or 90% of the full-length nucleicacid molecule of interest. Also contemplated are nucleic acid moleculesthat contain degenerate codons in place of codons in the hybridizingnucleic acid molecule. (For determination of homology of proteins,conservative amino acids can be aligned as well as identical aminoacids; in this case, percentage of identity and percentage homologyvary). Whether any two nucleic acid molecules have nucleotide sequences(or any two polypeptides have amino acid sequences) that are at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determinedusing known computer algorithms such as the “FAST A” program, using forexample, the default parameters as in Pearson et al. Proc. Natl. Acad.Sci. USA 85: 2444 (1988) (other programs include the GCG program package(Devereux, J., et al., Nucleic Acids Research 12(I): 387 (1984)),BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J. Molec. Biol. 215:403(1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press,San Diego (1994), and Carillo et al. SIAM J Applied Math 48: 1073(1988)). For example, the BLAST function of the National Center forBiotechnology Information database can be used to determine identity.Other commercially or publicly available programs include DNAStar“MegAlign” program (Madison, Wis.) and the University of WisconsinGenetics Computer Group (UWG) “Gap” program (Madison Wis.)). Percenthomology or identity of proteins and/or nucleic acid molecules can bedetermined, for example, by comparing sequence information using a GAPcomputer program (e.g., Needleman et al. J. Mol. Biol. 48: 443 (1970),as revised by Smith and Waterman (Adv. Appl. Math. 2: 482 (1981)).Briefly, a GAP program defines similarity as the number of alignedsymbols (i.e., nucleotides or amino acids) which are similar, divided bythe total number of symbols in the shorter of the two sequences. Defaultparameters for the GAP program can include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non identities)and the weighted comparison matrix of Gribskov et al. Nucl. Acids Res.14: 6745 (1986), as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps. Therefore, as used herein, the term “identity” represents acomparison between a test and a reference polypeptide or polynucleotide.In one non-limiting example, “at least 90% identical to” refers topercent identities from 90 to 100% relative to the referencepolypeptides. Identity at a level of 90% or more is indicative of thefact that, assuming for exemplification purposes a test and referencepolynucleotide length of 100 amino acids are compared, no more than 10%(i.e., 10 out of 100) of amino acids in the test polypeptide differsfrom that of the reference polypeptides. Similar comparisons can be madebetween a test and reference polynucleotides. Such differences can berepresented as point mutations randomly distributed over the entirelength of an amino acid sequence or they can be clustered in one or morelocations of varying length up to the maximum allowable, e.g., 10/100amino acid difference (approximately 90% identity). Differences aredefined as nucleic acid or amino acid substitutions, insertions ordeletions. At the level of homologies or identities above about 85-90%,the result should be independent of the program and gap parameters set;such high levels of identity can be assessed readily, often withoutrelying on software.

The term substantially identical or homologous or similar varies withthe context as understood by those skilled in the relevant art andgenerally means at least 60% or 70%, preferably means at least 80%, morepreferably at least 90%, and most preferably at least 95%, 96%, 97%,98%, 99% or greater identity. As used herein, substantially identical toa product means sufficiently similar so that the property of interest issufficiently unchanged so that the substantially identical product canbe used in place of the product.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis and high performance liquid chromatography (HPLC), usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound can, however, be amixture of stereoisomers or isomers. In such instances, furtherpurification might increase the specific activity of the compound.

As used herein equivalent, when referring to two sequences of nucleicacids, means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When equivalent is used inreferring to two proteins or peptides or other molecules, it means thatthe two proteins or peptides have substantially the same amino acidsequence with only amino acid substitutions (such as, but not limitedto, conservative changes) or structure and the any changes do notsubstantially alter the activity or function of the protein or peptide.When equivalent refers to a property, the property does not need to bepresent to the same extent (e.g., two peptides can exhibit differentrates of the same type of enzymatic activity), but the activities areusually substantially the same. Complementary, when referring to twonucleotide sequences, means that the two sequences of nucleotides arecapable of hybridizing, typically with less than 25%, 15% or 5%mismatches between opposed nucleotides. If necessary, the percentage ofcomplementarity will be specified. Typically the two molecules areselected such that they will hybridize under conditions of highstringency.

As used herein, a receptor refers to a molecule that has an affinity fora ligand. Receptors can be naturally-occurring or synthetic molecules.Receptors also can be referred to in the art as anti-ligands. As usedherein, the receptor and anti-ligand are interchangeable. Receptors canbe used in their unaltered state or bound to other polypeptides,including as homodimers. Receptors can be attached to, covalently ornoncovalently, or in physical contact with, a binding member, eitherdirectly or indirectly via a specific binding substance or linker.Examples of receptors, include, but are not limited to: antibodies, cellmembrane receptors surface receptors and internalizing receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells, or other materials), drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles.

As used herein, bind, bound and binding refer to the binding betweenatoms or molecules with a K_(d) in the range of 10⁻² to 10⁻¹⁵ mole/L,generally, 10⁻⁶ to 10⁻¹⁵, 10⁻⁷ to 10⁻¹⁵ and typically 10⁻⁸ to 10⁻¹⁵(and/or a K_(a) of 10⁵-10¹², 10⁷-10¹², 10⁸-10¹² L/mole).

As used herein, luminescence refers to the detectable electromagnetic(EM) radiation, generally, ultraviolet (UV), infrared (IR) or visible EMradiation that is produced when the excited product of an exergonicchemical process reverts to its ground state with the emission of light.Chemiluminescence is luminescence that results from a chemical reaction.Bioluminescence is chemiluminescence that results from a chemicalreaction using biological molecules (or synthetic versions or analogsthereof) as substrates and/or enzymes. Fluorescence is luminescence inwhich light of a visible color is emitted from a substance understimulation or excitation by light or other forms radiation such asultraviolet (UV), infrared (IR) or visible EM radiation.

As used herein, chemiluminescence refers to a chemical reaction in whichenergy is specifically channeled to a molecule causing it to becomeelectronically excited and subsequently to release a photon therebyemitting visible light. Temperature does not contribute to thischanneled energy. Thus, chemiluminescence involves the direct conversionof chemical energy to light energy.

As used herein, bioluminescence, which is a type of chemiluminescence,refers to the emission of light by biological molecules, particularlyproteins. The essential condition for bioluminescence is molecularoxygen, either bound or free in the presence of an oxygenase, aluciferase, which acts on a substrate, a luciferin. Bioluminescence isgenerated by an enzyme or other protein (luciferase) that is anoxygenase that acts on a substrate luciferin (a bioluminescencesubstrate) in the presence of molecular oxygen and transforms thesubstrate to an excited state, which, upon return to a lower energylevel releases the energy in the form of light.

As used herein, the substrates and enzymes for producing bioluminescenceare generically referred to as luciferin and luciferase, respectively.When reference is made to a particular species thereof, for clarity,each generic term is used with the name of the organism from which itderives such as, for example, click beetle luciferase or fireflyluciferase.

As used herein, luciferase refers to oxygenases that catalyze a lightemitting reaction. For instance, bacterial luciferases catalyze theoxidation of flavin mononucleotide (FMN) and aliphatic aldehydes, whichreaction produces light. Another class of luciferases, found amongmarine arthropods, catalyzes the oxidation of Cypridina (Vargula)luciferin and another class of luciferases catalyzes the oxidation ofColeoptera luciferin.

Thus, luciferase refers to an enzyme or photoprotein that catalyzes abioluminescent reaction (a reaction that produces bioluminescence). Theluciferases, such as firefly and Gaussia and Renilla luciferases areenzymes which act catalytically and are unchanged during thebioluminescence generating reaction. The luciferase photoproteins, suchas the aequorin photoprotein to which luciferin is non-covalently bound,are changed, such as by release of the luciferin, during bioluminescencegenerating reaction. The luciferase is a protein, or a mixture ofproteins (e.g., bacterial luciferase), that occurs naturally in anorganism or a variant or mutant thereof, such as a variant produced bymutagenesis that has one or more properties, such as thermal stability,that differ from the naturally-occurring protein. Luciferases andmodified mutant or variant forms thereof are well known. For purposesherein, reference to luciferase refers to either the photoproteins orluciferases.

Thus, reference, for example, to Renilla luciferase refers to an enzymeisolated from member of the genus Renilla or an equivalent moleculeobtained from any other source, such as from another related copepod, orthat has been prepared synthetically. It is intended to encompassRenilla luciferases with conservative amino acid substitutions that donot substantially alter activity. Conservative substitutions of aminoacids are known to those of skill in this art and can be made generallywithout altering the biological activity of the resulting molecule.Those of skill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. co., p. 224).

As used herein, bioluminescence substrate refers to the compound that isoxidized in the presence of a luciferase and any necessary activatorsand generates light. These substrates are referred to as luciferinsherein, are substrates that undergo oxidation in a bioluminescencereaction. These bioluminescence substrates include any luciferin oranalog thereof or any synthetic compound with which a luciferaseinteracts to generate light. Typical substrates include those that areoxidized in the presence of a luciferase or protein in alight-generating reaction. Bioluminescence substrates, thus, includethose compounds that those of skill in the art recognize as luciferins.Luciferins, for example, include firefly luciferin, Cypridina (alsoknown as Vargula) luciferin (coelenterazine), bacterial luciferin aswell as synthetic analogs of these substrates or other compounds thatare oxidized in the presence of a luciferase in a reaction the producesbioluminescence.

As used herein, capable of conversion into a bioluminescence substraterefers to being susceptible to chemical reaction, such as oxidation orreduction, which yields a bioluminescence substrate. For example, theluminescence producing reaction of bioluminescent bacteria involves thereduction of a flavin mononucleotide group (FMN) to reduced flavinmononucleotide (FMNH₂) by a flavin reductase enzyme. The reduced flavinmononucleotide (substrate) then reacts with oxygen (an activator) andbacterial luciferase to form an intermediate peroxy flavin thatundergoes further reaction, in the presence of a long-chain aldehyde, togenerate light. With respect to this reaction, the reduced flavin andthe long chain aldehyde are bioluminescence substrates.

As used herein, a bioluminescence generating system refers to the set ofreagents required to conduct a bioluminescent reaction. Thus, thespecific luciferase, luciferin and other substrates, solvents and otherreagents that can be required to complete a bioluminescent reaction forma bioluminescence system. Thus a bioluminescence generating systemrefers to any set of reagents that, under appropriate reactionconditions, yield bioluminescence. Appropriate reaction conditions referto the conditions necessary for a bioluminescence reaction to occur,such as pH, salt concentrations and temperature. In general,bioluminescence systems include a bioluminescence substrate, luciferin,a luciferase, which includes enzymes luciferases and photoproteins andone or more activators. A specific bioluminescence system can beidentified by reference to the specific organism from which theluciferase derives; for example, the Renilla bioluminescence systemincludes a Renilla luciferase, such as a luciferase isolated fromRenilla or produced using recombinant methods or modifications of theseluciferases. This system also includes the particular activatorsnecessary to complete the bioluminescence reaction, such as oxygen and asubstrate with which the luciferase reacts in the presence of the oxygento produce light.

As used herein, a fluorescent protein (FP) refers to a protein thatpossesses the ability to fluoresce (i.e., to absorb energy at onewavelength and emit it at another wavelength). For example, a greenfluorescent protein (GFP) refers to a polypeptide that has a peak in theemission spectrum at 510 nm or about 510 nm. A variety of FPs that emitat various wavelengths are known in the art. Exemplary FPs include, butare not limited to, a green fluorescent protein (GFP), yellowfluorescent protein (YFP), orange fluorescent protein (OFP), cyanfluorescent protein (CFP), blue fluorescent protein (BFP), redfluorescent protein (RFP), far-red fluorescent protein, or near-infraredfluorescent protein. Extending the spectrum of available colors offluorescent proteins to blue, cyan, orange yellow and red variants,provides a method for multicolor tracking of fusion proteins.

As used herein, Aequorea GFP refers to GFPs from the genus Aequorea andto mutants or variants thereof. Such variants and GFPs from otherspecies, such as Anthozoa reef coral, Anemonia sea anemone, Renilla seapansy, Galaxea coral, Acropora brown coral, Trachyphyllia and Pectimidaestony coral and other species are well known and are available and knownto those of skill in the art. Exemplary GFP variants include, but arenot limited to BFP, CFP, YFP and OFP. Examples of florescent proteinsand their variants include GFP proteins, such as Emerald (InVitrogen,Carlsbad, Calif.), EGFP (Clontech, Palo Alto, Calif.), Azami-Green (MBLInternational, Woburn, Mass.), Kaede (MBL International, Woburn, Mass.),ZsGreenl (Clontech, Palo Alto, Calif.) and CopGFP (Evrogen/Axxora, LLC,San Diego, Calif.); CFP proteins, such as Cerulean (Rizzo, Nat.Biotechnol. 22(4):445-9 (2004)), mCFP (Wang et al., PNAS USA.101(48):16745-9 (2004)), AmCyan1 (Clontech, Palo Alto, Calif.), MiCy(MBL International, Woburn, Mass.), and CyPet (Nguyen and Daugherty,Nat. Biotechnol. 23(3):355-60 (2005)); BFP proteins such as EBFP(Clontech, Palo Alto, Calif.); YFP proteins such as EYFP (Clontech, PaloAlto, Calif.), YPet (Nguyen and Daugherty, Nat. Biotechnol. 23(3):355-60(2005)), Venus (Nagai et al., Nat. Biotechnol. 20(1):87-90 (2002)),ZsYellow (Clontech, Palo Alto, Calif.), and mCitrine (Wang et al., PNASUSA. 101(48): 16745-9 (2004)); OFP proteins such as cOFP (Strategene, LaJolla, Calif.), mKO (MBL International, Woburn, Mass.), and mOrange; andothers (Shaner N C, Steinbach P A, and Tsien R Y., Nat. Methods.2(12):905-9 (2005)).

As used herein, red fluorescent protein, or RFP, refers to the DiscosomaRFP (DsRed) that has been isolated from the corallimorph Discosoma (Matzet al., Nature Biotechnology 17: 969-973 (1999)), and red or far-redfluorescent proteins from any other species, such as Heteractis reefcoral and Actinia or Entacmaea sea anemone, as well as variants thereof.RFPs include, for example, Discosoma variants, such as mRFP1, mCherry,tdTomato, mStrawberry, mTangerine (Wang et al., PNAS USA. 101(48):16745-9 (2004)), DsRed2 (Clontech, Palo Alto, Calif.), and DsRed-T1(Bevis and Glick, Nat. Biotechnol., 20: 83-87 (2002)), Anthomedusa J-Red(Evrogen) and Anemonia AsRed2 (Clontech, Palo Alto, Calif.). Far-redfluorescent proteins include, for example, Actinia AQ143 (Shkrob et al.,Biochem J 392(Pt 3):649-54 (2005)), Entacmaea eqFP611 (Wiedenmann et al.Proc Natl Acad Sci USA. 99(18):11646-51 (2002)), Discosoma variants suchas mPlum and mRasberry (Wang et al., PNAS USA. 101(48):16745-9 (2004)),and Heteractis HcRed1 and t-HcRed (Clontech, Palo Alto, Calif.).

As used herein the term assessing or determining is intended to includequantitative and qualitative determination in the sense of obtaining anabsolute value for the activity of a product, and also of obtaining anindex, ratio, percentage, visual or other value indicative of the levelof the activity. Assessment can be direct or indirect.

As used herein, activity refers to the in vivo activities of a compoundor viruses on physiological responses that result following in vivoadministration thereof (or of a composition or other mixture). Activity,thus, encompasses resulting therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Activities can beobserved in in vitro and/or in vivo systems designed to test or use suchactivities.

As used herein, a vaccine refers to a composition which, uponadministration to a subject, elicits an immune response in a subject towhich it is administered and which protects the immunized subjectagainst subsequent challenge by the immunizing agent or animmunologically cross-reactive agent. A vaccine can be used to enhancethe immune response against a pathogen, such as a virus, that expressesthe immunological agent and/or has already infected the subject.Protection can be complete or partial (i.e., a reduction in symptoms orinfection as compared with an unvaccinated subject). Typically a vaccineis administered to a subject that is a mammal. An immunologicallycross-reactive agent can be, for example, the whole protein (e.g., tumorantigen) from which a subunit peptide used as the immunogen is derived.Alternatively, an immunologically cross-reactive agent can be adifferent protein which is recognized in whole or in part by theantibodies elicited by the immunizing agent. Exemplary vaccines can bemodified vaccinia viruses that express an immunologically cross-reactiveagent.

As used herein, a “pharmaceutically acceptable carrier” refers to anycarrier, diluent, excipient, wetting agent, buffering agent, suspendingagent, lubricating agent, adjuvant, solid binder, vehicle, deliverysystem, emulsifier, disintegrant, absorbent, preservative, surfactant,colorant, flavorant, or sweetener, preferably non-toxic, that aresuitable for use in a pharmaceutical composition.

As used herein, complex refers generally to an association between twoor more species regardless of the nature of the interaction between thespecies (i.e., ionic, covalent, or electrostatic).

As used herein, “a combination” refers to any association between two oramong more items or elements. Exemplary combinations include, but arenot limited to, two or more pharmaceutical compositions, a compositioncontaining two or more active ingredients, such as two viruses, or avirus and a chemotherapeutic compound, two or more viruses, a virus anda therapeutic agent, a virus and an imaging agent, a virus and aplurality therapeutic and/or imaging agents, or any association thereof.Such combinations can be packaged as kits.

As used herein, a composition refers to any mixture. It can be asolution, a suspension, an emulsion, liquid, powder, a paste, aqueous,non-aqueous or any combination of such ingredients.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, a kit is a packaged combination, optionally, includinginstructions for use of the combination and/or other reactions andcomponents for such use.

For clarity of disclosure, and not by way of limitation, the detaileddescription is divided into the subsections that follow.

B. VIRUSES FOR TREATMENT AND DIAGNOSIS

Provided herein are viruses for therapeutic and diagnostic use. Alsoprovided elsewhere herein are methods for making and using such virusesfor therapeutic and diagnostic use. The viruses provided herein aretypically attenuated. Attenuated viruses have a decreased capacity tocause disease in a host. The decreased capacity can result from any of avariety of different modifications to the ability of a virus to bepathogenic. For example, a virus can have reduced toxicity, reducedability to accumulate in non-tumorous organs or tissue, reduced abilityto cause cell lysis or cell death, or reduced ability to replicatecompared to the non-attenuated form thereof. The attenuated virusesprovided herein, however, retain at least some capacity to replicate andto cause immunoprivileged cells and tissues, such as tumor cells to leakor lyse, undergo cell death, or otherwise cause or enhance an immuneresponse to immunoprivileged cells and tissues, such as tumor cells.Such characteristics of the viruses provided are described in detailelsewhere herein.

The viruses provided herein can accumulate in immunoprivileged cells orimmunoprivileged tissues, including tumors and/or metastases, and alsoincluding wounded tissues and cells. While the viruses provided hereincan typically be cleared from the subject to whom the viruses areadministered by activity of the subject's immune system, viruses cannevertheless accumulate, survive and proliferate in immunoprivilegedcells and tissues such as tumors because such immunoprivileged areas aresequestered from the host's immune system. Accordingly, the methodsprovided herein, as applied to tumors and/or metastases, and therapeuticmethods relating thereto, can readily be applied to otherimmunoprivileged cells and tissues, including wounded cells and tissues.

Among the viruses provided herein are cytoplasmic viruses, which do notrequire entry of viral nucleic acid molecules in to the nucleus of thehost cell during the viral life cycle. Exemplary cytoplasmic virusesprovided herein are viruses of the poxvirus family, includingorthopoxviruses. Exemplary of poxviruses provided herein are vacciniaviruses. Vaccinia virus possesses a variety of features for use incancer gene therapy and vaccination, including broad host and cell typerange, a large carrying capacity for foreign genes and high sequencehomology among different strains for designing and generating modifiedviruses in other strains. Techniques for production of modified vacciniastrains by genetic engineering are well established (Moss (1993) Curr.Opin. Genet. Dev. 3: 86-90; Broder and Earl (1999) Mol. Biotechnol. 13:223-245; Timiryasova et al. (2001) Biotechniques 31: 534-540). A varietyof vaccinia virus strains are available, including Western Reserve (WR),Copenhagen, Tashkent, Tian Tan, Lister, Wyeth, 1HD-J, and IHD-W,Brighton, Ankara, MVA, Dairen I, LIPV, LC16M8, LC16MO, LIVP, WR 65-16,Connaught, New York City Board of Health. Exemplary of vaccinia virusesprovided herein are Lister strain or LIVP vaccinia viruses.

The viruses provided herein are modified from their wild type form.Modifications can include any of a variety of changes, and includechanges to the genome of the virus. Exemplary nucleic acid modificationsinclude truncations, insertions, deletions and mutations. In anexemplary modification, a viral gene can be modified by truncation,insertion, deletion or mutation. Modifications of the viruses providedherein can result in a modification of virus characteristics, includingthose provided herein such as pathogenicity, toxicity, ability topreferentially accumulate in tumor, ability to lyse cells or cause celldeath, ability to elicit an immune response against tumor cells,immunogenicity and replication competence.

Provided herein are vaccinia viruses with insertions, mutations ordeletions, as provided in the Examples and described elsewhere herein.Exemplary insertions, mutations or deletions are those that result in anattenuated vaccinia virus relative to the wild type strain. For example,vaccinia virus insertions, mutations or deletions can decreasepathogenicity of the vaccinia virus, for example, by reducing thetoxicity, reducing the infectivity, reducing the ability to replicate orreducing the number of non-tumor organs or tissues to which the vacciniavirus can accumulate. Other exemplary insertions, mutations or deletionsinclude, but are not limited to, those that increase antigenicity of thevirus, those that permit detection or imaging, those that alterattenuation of the virus, and those that alter infectivity.Modifications can be made, for example, in genes that are involved innucleotide metabolism, host interactions and virus formation.

Any of a variety of insertions, mutations or deletions of the vacciniavirus known in the art can be used herein, including insertions,mutations or deletions of: the thymidine kinase (TK) gene, thehemagglutinin (HA) gene and F14.5L gene, among others provided elsewhereherein. The vaccinia viruses provided herein also can contain two ormore insertions, mutations or deletions. Thus, included are vacciniaviruses containing two or more insertions, mutations or deletions of theloci provided herein or other loci known in the art.

Viruses provided herein can contain one or more heterologous nucleicacid molecules inserted into the genome of the virus. A heterologousnucleic acid molecule can contain an open reading frame or can be anon-coding sequence. In some cases, the heterologous nucleic acidreplaces all or a portion of a viral gene. The viral gene can bereplaced with homologous gene from another virus or a different gene.For example, vaccinia viruses provided herein can be modified byreplacement of the A34R gene with another A34R gene from a differentstrain in order to increase the EEV form of the virus. In one example,the A34R gene from the Lister strain of vaccinia can be replaced withA34R gene from the IHD-J strain of vaccinia virus (see, e.g., Examples1, 2; strain GLV-1i69).

The heterologous nucleic acid can be operably linked to a promoter forexpression of an open reading frame. A heterologous nucleic acid that isoperably linked to a promoter is also called an expression cassette.Hence, viruses provided herein can have the ability to express one ormore heterologous genes. Gene expression can include expression of aprotein encoded by a gene and/or expression of an RNA molecule encodedby a gene. In some embodiments, the viruses provided herein can expressexogenous genes at levels high enough that permit harvesting products ofthe exogenous genes from the tumor. Expression of heterologous genes canbe controlled by a constitutive promoter, or by an inducible promoter.Exogenous genes expressed can include genes encoding a therapeutic geneproduct, genes encoding a detectable gene product such as a gene productthat can be used for imaging, genes encoding a gene product to beharvested, genes encoding an antigen for tumor therapy or for antibodyto be harvested (e.g., vaccination). The viruses provided herein can beused for expressing genes in vivo and in vitro.

The heterologous gene expressed by the viruses provided herein can becontrolled by a regulatory sequence. Suitable regulatory sequenceswhich, for example, are functional in a mammalian host cell are wellknown in the art. In one example, the regulatory sequence can contain anatural or synthetic promoter. In another embodiment, the regulatorysequence contains a poxvirus promoter, such as a vaccinia viruspromoter. Strong late promoters can be used to achieve high levels ofexpression of the foreign genes. Early and intermediate-stage promoterscan also be used. In one embodiment, the promoters contain early andlate promoter elements, for example, the vaccinia virus early/latepromoter P7.5 k, vaccinia late promoter P11 k, a synthetic early/latevaccinia PSEL promoter (Patel et al., (1988) Proc. Natl. Acad. Sci. USA85: 9431-9435; Davison and Moss, (1989) J Mol Biol 210: 749-769; Davisonet al. (1990) Nucleic Acids Res. 18: 4285-4286; Chakrabarti et al.(1997), BioTechniques 23: 1094-1097). As described in the Examples andelsewhere herein, the viruses provided can exhibit differences incharacteristics, such as attenuation, as a result of using a strongerpromoter versus a weaker promoter. For example, in vaccinia, syntheticearly/late and late promoters are relatively strong promoters, whereasvaccinia synthetic early, P7.5 k early/late, P7.5 k early, and P28 latepromoters are relatively weaker promoters (see e.g., Chakrabarti et al.(1997) BioTechniques 23(6) 1094-1097).

The viruses provided herein can express one or more genes whose productsare useful for tumor therapy. For example, a virus can express aproteins cause cell death or whose products cause an anti-tumor immuneresponse. Such genes can be considered therapeutic genes. A variety oftherapeutic gene products, such as toxic or apoptotic proteins, orsiRNA, are known in the art, and can be used with the viruses providedherein. The therapeutic genes can act by directly killing the host cell,for example, as a channel-forming or other lytic protein, or bytriggering apoptosis, or by inhibiting essential cellular processes, orby triggering an immune response against the cell, or by interactingwith a compound that has a similar effect, for example, by converting aless active compound to a cytotoxic compound. Exemplary proteins usefulfor tumor therapy include, but are not limited to, tumor suppressors,toxins, cytostatic proteins and costimulatory molecules, such ascytokines and chemokines among others provided elsewhere herein andknown in the art.

The viruses provided herein can be based on modifications to the Listerstrain of vaccinia virus (e.g., LIVP). The modifications of the Listerstrain provided herein can also be adapted to other vaccinia viruses(e.g., Western Reserve (WR), Copenhagen, Tashkent, Tian Tan, Lister,Wyeth, 1HD-J, and IHD-W, Brighton, Ankara, MVA, Dairen I, LIPV, LC16M8,LC16MO, LIVP, WR 65-16, Connaught, New York City Board of Health). Themodifications of the Lister strain provided herein can also be adaptedto other viruses, including, but not limited to, viruses of the poxvirusfamily, adenoviruses, herpes viruses and retroviruses.

Exemplary vaccinia viruses provided herein were derived from vacciniavirus strain GLV-1h68 (also named RVGL21, SEQ ID NO: 1). GLV-1h68, whichhas been described in U.S. Pat. Pub. No. 2005-0031643, contains DNAinsertions gene loci of the vaccinia virus LIVP strain (a vaccinia virusstrain, originally derived by adapting the Lister strain (ATCC CatalogNo. VR-1549) to calf skin (Institute of Viral Preparations, Moscow,Russia, Al'tshtein et al., (1983) Dokl. Akad. Nauk USSR 285:696-699)).GLV-1h68 contains expression cassettes encoding detectable markerproteins in the F14.5L (also designated in LIVP as F3) gene locus,thymidine kinase (TK) gene locus, and hemagglutinin (HA) gene locus. Anexpression cassette containing a Ruc-GFP cDNA molecule (a fusion of DNAencoding Renilla luciferase and DNA encoding GFP) under the control of avaccinia synthetic early/late promoter P_(SEL) ((P_(SEL))Ruc-GFP) wasinserted into the F14.5L gene locus; an expression cassette containing aDNA molecule encoding beta-galactosidase under the control of thevaccinia early/late promoter P_(7.5k) ((P_(7.5k))LacZ) and DNA encodinga rat transferrin receptor positioned in the reverse orientation fortranscription relative to the vaccinia synthetic early/late promoterP_(SEL) ((P_(SEL))rTrfR) was inserted into the TK gene locus (theresulting virus does not express transferrin receptor protein since theDNA molecule encoding the protein is positioned in the reverseorientation for transcription relative to the promoter in the cassette);and an expression cassette containing a DNA molecule encodingβ-glucuronidase under the control of the vaccinia late promoter P_(11k)((P_(11k))gusA) was inserted into the HA gene locus.

1. Viruses with Altered Infectivity

Provided herein are modifications of vaccinia viruses that alter theability of the viruses to infect and replicate within tumors.Infectivity can be enhanced by modification of viral coat proteins thatare involved in cellular in infection or are targeted by the host immunesystem. Coat proteins, such as that A34R protein, affect sensitivity ofthe virus to complement and/or antibody neutralization. Exemplarymodifications in coat proteins include mutations or replacement of viralcoat proteins, which can increase production of resistant viral forms inby host cell. Also provided herein are modifications that increase ordecrease the transcriptional and/or translational load on the virus.Exemplary modifications include insertion and/or deletion of geneexpression cassettes or replacement of genes with non-codingheterologous nucleic acid, which increase or decreases the number oftranscriptional/translational units carried by the virus.

a. Viruses with Modified Viral Proteins

i. Increase in the Vaccinia EEV Form by Replacement of A34R

Vaccinia virus replicates in cells and produces both intracellular virus(IMV, intracellular mature virus; IEV, intracellular enveloped virus)and extracellular virus (EEV, extracellular enveloped virus; CEV,cell-associated extracellular virus) (Smith et al. (1998) Adv Exp Med.Biol. 440: 395-414). IMV represents approximately 99% of virus yieldfollowing replication by wild-type vaccinia virus strains. The IMV virusform is relatively stable in the outside environment, and is primarilyresponsible for spread between individuals; however, IMV virus does notspread efficiently within the infected host due to inefficient releasefrom cells and sensitivity to complement and/or antibody neutralization.By contrast, the EEV form is released into the extracellular milieu andtypically represents only approximately 1% of the viral yield (Smith etal. (1998) Adv Exp Med. Biol. 440: 395-414). EEV is responsible forviral spread within the infected host and is relatively easily degradedoutside of the host. In addition, the EEV form has developed severalmechanisms to inhibit its neutralization within the bloodstream. EEV isrelatively resistant to complement (Vanderplasschen et al. (1998) ProcNatl Acad Sci USA. 95(13): 7544-9) due to the incorporation of host cellinhibitors of complement into its outer membrane coat and secretion ofvaccinia virus complement control protein (VCP) into local extracellularenvironment. In addition, EEV is relatively resistant to neutralizingantibody effects compared to IMV (Smith et al. (1997) Immunol Rev. 159:137-54; Vanderplasschen et al. (1997) J Gen Virol. 78 (Pt 8): 2041-8).EEV is released at earlier time points following infection (e.g., 4-6hours) than is IMV (which is only released during/after cell death), andtherefore, spread of the EEV form is faster (Blasco et al. (1993) JVirol. 67(6):3319-25).

The EEV form of vaccinia virus has naturally acquired features for rapidand efficient spread through solid tumors locally and to regional ordistant tumor sites. Since EEV is relatively resistant to complementeffects and to antibody-mediated neutralization, when it is grown in acell type from the same species, this virus form will have enhancedstability and retain activity longer in the blood followingintravascular administration (Smith et al. (1998) Adv Exp Med Biol. 440:395-414; Vanderplasschen et al., (1998) Proc Natl Acad Sci USA.(13):7544-9). This is particularly important for repeat administrationonce neutralizing antibody levels have increased and anti-cancertherapies require repeat administration. Therefore, increasing the EEVform of vaccinia, and other poxviruses, results in enhanced systemicefficacy. Polypeptides involved in the modulation of the EEV form of apoxvirus include, but are not limited to, A34R and B5R. A mutation atcodon 151 of A34R from a lysine to an aspartic acid K151D mutationrenders the A34R protein less able to tether the EEV form to the cellmembrane. B5R is an EEV-membrane bound polypeptide that can bindcomplement. The total deletion of A43R can lead to increased EEVrelease, but markedly reduced infectivity of the viruses, while theK151D mutation increases EEV release while maintaining infectivity ofthe released viruses.

The ability of vaccinia viruses provided herein to infect and replicatewithin tumors can be enhanced by increasing the extracellular envelopedform of the virus (EEV). The methods provided herein for modulating theattenuation of a virus can be combined with any known method forincreasing the EEV form of the virus. For example, vaccinia virusesprovided herein can be modified by replacement of the A34R gene withanother A34R gene from a different strain. In one example, the A34R genefrom the Lister strain of vaccinia can be replaced with A34R gene fromthe IHD-J strain of vaccinia virus (see e.g., Examples 1, 2; strainGLV-1i69). A34R gene from the IHD-J strain contains a mutation thatincreases the percentage of EEV form of the virus. In another example,the A34R gene of the vaccinia viruses provided herein can also bemutated to increase the amount of EEV particles released.

ii. Deletion of A35R

Modification of viral proteins can also be employed to attenuate theviruses. Deletion of genes encoding viral proteins, such as A35R, candecrease the toxicity of vaccinia strains (Roper, R. L. (2006) J. Virol.80(1) 306-313). The A35R deletion can attenuate toxicity of the viruswhen injected into mice without affecting viral properties, such asviral plaque size, viral replication, host range or viralinfectivity/spread. Provided herein are viruses that have the A35R genedeleted (see e.g., Examples 1, 16; strains GLV-1j87, GLV-1j88 andGLV-1j89).

b. Viruses with Multiple Genome Insertions and/or Deletions

As described in the Examples, viruses provided herein can exhibitdifferences in characteristics, such as attenuation, as a result ofinserting one or more expression cassettes into the viral genome,removing one or more expression cassettes from the viral genome orreplacing one or more expression cassettes in the viral genome. Forexample, a decrease in attenuation was observed when one or moreexpression cassettes was removed from a viral genome, such as the viralgenome of the recombinant vaccinia LIVP strain GLV-1h68. In someexamples, vaccinia viruses provided herein can have one or moreexpression cassettes removed from a virus and replaced with aheterologous non-coding nucleic acid molecule (see, e.g., strainsGLV-1h70, GLV-1h71, GLV-1h72, GLV-1h73, GLV-1h74, GLV-1h85, andGLV-1h86). In other examples, vaccinia viruses provided herein can haveone or more expression cassettes removed from a virus and replaced witha heterologous nucleic acid molecule that encodes a polypeptide (see,e.g., strains GLV-1h81, GLV-1h82, GLV-1h83, GLV-1h84, GLV-1h84,GLV-1h90, GLV-1h91, GLV-1h92, GLV-1h96, GLV-1h97, GLV-1h98, GLV-1h104,GLV-1h105, GLV-1h106, GLV-1h107, GLV-1h108 and GLV-1h109).

Vaccinia viruses are provided herein that differ in the level ofattenuation exhibited by the virus in vivo and in vitro. As described inthe Examples and elsewhere herein, the level of attenuation was modifiedby altering the number of expression cassettes contained in the virus orby modifying one or more expression cassettes contained in the virus byremoval or replacement. Such modifications can increase or decrease thetranscriptional or translation load on the virus, resulting in analtered level of attenuation.

Vaccinia viruses provided herein can have one or more expressioncassettes removed from GLV-1h68 and replaced with a heterologousnon-coding DNA molecule. Exemplary viruses provided include GLV-1h70,GLV-1h71, GLV-1h72, GLV-1h73, GLV-1h74, GLV-1h85, and GLV-1h86. GLV-1h70contains (P_(SEL))Ruc-GFP inserted into the F14.5L gene locus,(P_(SEL))rTrfR and (P_(7.5k))LacZ inserted into the TK gene locus, and anon-coding DNA molecule inserted into the HA gene locus in place of(P_(11k))gusA. GLV-1h71 contains a non-coding DNA molecule inserted intothe F14.5L gene locus in place of (P_(SEL))Ruc-GFP, (P_(SEL))rTrfR and(P_(7.5k))LacZ inserted into the TK gene locus, and (P_(11k))gusAinserted into the HA gene locus. GLV-1h72 contains (P_(SEL))RUC-GFPinserted into the F14.5L gene locus, a non-coding DNA molecule insertedinto the TK gene locus in place of (P_(SEL))rTrfR and (P_(7.5k))LacZ,and P_(11k)gusA inserted into the HA gene locus. GLV-1h73 contains anon-coding DNA molecule inserted into the F14.5L gene locus in place of(P_(SEL))Ruc-GFP, (P_(SEL))rTrfR and (P_(7.5k))LacZ inserted into the TKgene locus, and a non-coding DNA molecule inserted into the HA genelocus in place of (P_(11k))gusA. GLV-1h74 contains a non-coding DNAmolecule inserted into the F14.5L gene locus in place of(P_(SEL))RUC-GFP, a non-coding DNA molecule inserted into the TK genelocus in place of (P_(SEL))rTrfR and (P_(7.5k))LacZ, and a non-codingDNA molecule inserted into the HA gene locus in place of (P_(11k))gusA.GLV-1h85 contains a non-coding DNA molecule inserted into the F14.5Lgene locus in place of (P_(SEL))RUC-GFP, a non-coding DNA moleculeinserted into the TK gene locus in place of (P_(SEL))rTrfR and(P_(7.5k))LacZ, and (P_(11k))gusA inserted into the HA gene locus.GLV-1h86 contains (P_(SEL))RUC-GFP inserted into the F14.5L gene locus,a non-coding DNA molecule inserted into the TK gene locus in place of(P_(SEL))rTrfR and (P_(7.5k))LacZ, and a non-coding DNA moleculeinserted into the HA gene locus in place of (P_(11k))gusA.

2. Viruses that Express Proteins for Tumor Imaging

The viruses provided herein can express one or more genes whose productsare detectable or whose products can provide a detectable signal. Avariety of detectable gene products, such as detectable proteins areknown in the art, and can be used with the viruses provided herein.Detectable proteins include receptors or other proteins that canspecifically bind a detectable compound, proteins that can emit adetectable signal such as a fluorescence signal, or enzymes that cancatalyze a detectable reaction or catalyze formation of a detectableproduct.

A variety of DNA sequences encoding proteins that can emit a detectablesignal or that can catalyze a detectable reaction, such as luminescentor fluorescent proteins, are known and can be used in the virus andmethods provided herein. Exemplary detectable gene products aredescribed else where herein and include, but are not limited to fireflyluciferase (de Wet et al. (1987) Mol. Cell. Biol. 7: 725-737), Renillaluciferase from Renilla renformis (Lorenz et al. (1991) PNAS USA 88:4438-4442), click beetle luciferase (CBG99; Wood et al. (1989) Science244(4905): 700-2), green fluorescent protein from Aequorea victoria(Prasher et al. (1987) Gene 111: 229-233) and red fluorescent from thecorallimorph Discosoma (Matz et al. (1999) Nature Biotechnology 17:969-973). Additional detectable proteins include reporter proteins, suchas E. coli β galactosidase (LacZ), β glucuronidase (gusA),xanthineguanine phosphoribosyltransferase (XGPRT).

In some examples, two or more detectable proteins are fused together toproduce a single polypeptide. Provided herein are viruses that contain agene encoding a Renilla luciferase fused to a green fluorescent protein,Ruc-GFP. Exemplary viruses include, but are not limited to, GLV-1h68,GLV-1i69, GLV-1j87, GLV-1h70, GLV-1h72, GLV-1h82, GLV-1h83, GLV-1h86,GLV-1h90, GLV-1h91, GLV-1h92, GLV-1h96, GLV-1h97, GLV-1h98, GLV-1h104,GLV-1h105, GLV-1h106, GLV-1h107, GLV-1h108 and GLV-1h109. These virusescontain an insertion of an expression cassette into the F14.5L genelocus, where the expression cassette encodes Ruc-GFP under the controlof a vaccinia synthetic early/late promoter P_(SEL).

In some examples, two or more detectable proteins are produced from asingle transcript that produces two separate polypeptides duringtranslation. Provided herein are viruses that contain a DNA encoding aclick beetle luciferase (CBG99) and monomeric red fluorescent protein(mRFP1) connected through a picornavirus 2A element (e.g., GLV-1h84).During translation, the two proteins are cleaved into two individualproteins at the picornavirus 2A element (Osborn et al., Mol. Ther. 12:569-74, 2005). GLV-1h84 contains an insertion of an expression cassetteinto the F14.5L gene locus, where the expression cassette encodesRuc-GFP under the control of a vaccinia synthetic early/late promoterP_(SEL).

A variety of gene products, such as proteins, that can specifically binda detectable compound are known in the art, including receptors (e.g.,transferrin receptor), metal binding proteins (e.g., ferritin), ligandbinding proteins and antibodies. Any of a variety of detectablecompounds can be used, and can be imaged by any of a variety of knownimaging methods. Exemplary compounds include receptor ligands andantigens for antibodies. The ligand can be labeled according to theimaging method to be used. Exemplary imaging methods include any of avariety magnetic resonance methods, such as magnetic resonance imaging(MRI) and magnetic resonance spectroscopy (MRS), and also include any ofa variety of tomographic methods, such as positron emission tomography(PET). An exemplary virus provided herein that expresses a protein thatcan bind a detectable compound is a vaccinia virus that expresses aferritin. GLV-1h82 and GLV-1h83 contain an insertion of an expressioncassette into the HA gene locus where the expression cassette encodes aferritin from E. coli under the control of a vaccinia syntheticearly/late promoter P_(SEL). An exemplary virus provided herein thatexpresses a protein that can bind a detectable compound is a vacciniavirus that expresses a transferrin receptor. GLV-1h82 additionallycontains an insertion of an expression cassette into the TK gene locuswhere the expression cassette encodes a transferrin receptor under thecontrol of a vaccinia synthetic early/late promoter P_(SEL).

3. Viruses that Express Proteins for Tumor Treatment

Viruses provided herein can express one or more therapeutic geneproducts. Such proteins can inhibit tumor growth or whose products causean anti-tumor immune response. Among the vaccinia viruses providedherein are vaccinia viruses that express protein for inhibition ofangiogenesis and/or suppression of tumor cell growth. Particular virusesthat express therapeutic gene products Lister strain vaccinia viruses.Exemplary Lister strain vaccinia viruses are provided here and describedelsewhere herein.

a. Proteins for Inhibiting Angiogenesis

Among the vaccinia viruses provided herein are vaccinia viruses thatexpress protein for inhibition of blood vessel formation. Inhibition ofangiogenesis promotes inhibition of tumor growth by inhibitingvascularization of the tumor needed to for the expansion of the tumormass.

i. hk5

In one example, viruses provided herein are modified to express theplasminogen K5 domain. Plasminogen kringle 5 is a potent angiogenesisinhibitor, which has been shown to induce apoptosis of endothelial cellsand inhibit their migration. Human plasminogen kringle 5 has also beenshown to induce apoptosis of tumor cells (Davidson et al. (2005) CancerRes. 65: 4663-4672). Exemplary vaccinia viruses that express theplasminogen K5 domain under the control of the vaccinia syntheticearly-late promoter are provided herein and described in further detailin the Examples (e.g., GLV-1h71).

ii. tTF-RGD

In one example, viruses provided herein are modified to express a fusionprotein containing a truncated human tissue factor protein fused to anRGD peptide (tTF-RGD). The fusion protein binds selectively to tumorvessel endothelial cells via the RGD peptide portion. The tissue factoris able to activate blood clotting once bound to the tumor vesselendothelial cells, which in turn inhibits neovascularization of thetumor. Vaccinia viruses provided herein can effect tumor localizedexpression of tTF-RGD for the inhibition of tumor vascularization.Exemplary vaccinia viruses that express tTF-RGD under the control of avaccinia synthetic early promoter, vaccinia synthetic early/latepromoter or vaccinia synthetic late promoter are provided herein anddescribed in further detail in the Examples (e.g., GLV-1h104, GLV-1h105and GLV-1h106).

iii. Anti-VEGF scAb

In one example, viruses provided herein are modified to express a fusionprotein containing a single chain anti-VEGF antibody fused to an FLAGpeptide (G6-FLAG). Vascular endothelial growth factor (VEGF) functionsas a major inducer of angiogenesis. Monoclonal antibodies directedagainst VEGF can inhibit tumor growth in mice and is effective ininhibiting tumor growth the treatment of cancer patients. Single-chainAb fragments (scFvs or scAb) derived from anti-VEGF antibodies are alsopotent inhibitors of vascularization have been shown to reduce thegrowth of subcutaneous tumors in nude mice (Vitaliti et al. (2000)Cancer Research 60, 4311-4314). Vaccinia viruses provided herein caneffect tumor localized expression of scAb VEGF antibodies for theinhibition of tumor vascularization. Exemplary vaccinia viruses thatexpress G6-FLAG under the control of a vaccinia synthetic earlypromoter, vaccinia synthetic early/late promoter or vaccinia syntheticlate promoter are provided herein and described in further detail in theExamples (e.g., GLV-1h107, GLV-1h108 and GLV-1h109).

c. Proteins for Tumor Growth Suppression

i. sIL-6R-IL-6

In one example, viruses provided herein are modified to express a fusionprotein containing an IL-6 fused to an IL-6 receptor (sIL-6R/IL-6). ThesIL-6R/IL-6 fusion polypeptide is an effective in suppressor of tumorcell growth (see e.g., U.S. Pat. No. 7,112,436; U.S. Patent ApplicationSerial No. 2007-0172455; Özbek et al. (2001) Oncogene 20(8): 972-979).Vaccinia viruses provided herein can effect tumor localized expressionof sIL-6R-IL-6 for the inhibition of tumor cell growth. Exemplaryvaccinia viruses that express sIL-6R-IL-6 under the control of avaccinia synthetic early promoter, vaccinia synthetic early/latepromoter or vaccinia synthetic late promoter are provided herein anddescribed in further detail in the Examples (e.g., GLV-1h90, GLV-1h91and GLV-1h92).

ii. IL-24

In one example, viruses provided herein are modified to expressinterleukin-24 (IL-24). IL-24, also called, mda-7 or melanomadifferentiation gene is a potent inhibitor of tumor cell growth (seee.g., U.S. Pat. No. 5,710,137 and U.S. Patent Application Serial No.2006-0134801). Vaccinia viruses provided herein can effect tumorlocalized expression of IL-24 for the inhibition of tumor cell growth.Exemplary vaccinia viruses that express IL-24 under the control of avaccinia synthetic early promoter, vaccinia synthetic early/latepromoter or vaccinia synthetic late promoter are provided herein anddescribed in further detail in the Examples (e.g., GLV-1h96, GLV-1h97and GLV-1h98).

4. Viruses that Express Proteins for Combined Tumor Diagnosis andTreatment

Provided herein are viruses that express a detectable protein andtherapeutic protein. For example, viruses provided herein can express adetectable protein, such as Ruc-GFP fusion protein, and therapeuticprotein, such as protein for tumor therapy. Exemplary tumor therapeuticproteins expressed by viruses provided herein included, but are notlimited to, proteins that stimulate the host immune response (e.g., IL-6and IL-24) and a proteins that inhibit angiogenesis (e.g., tTF-RGD andanti-VEGF Abs). In some examples the detectable protein is Ruc-GFP andthe therapeutic protein is sIL-6R-IL-6 fusion protein (e.g., GLV-1h90,GLV-1h91 and GLV-1h92). In other examples, the detectable protein isRuc-GFP and the therapeutic protein is IL-24 (e.g., GLV-1h96, GLV-1h97and GLV-1h98). In other examples, the detectable protein is Ruc-GFP andthe therapeutic protein is tTF-RGD fusion protein (e.g., GLV-1h104,GLV-1h105 and GLV-1h106). In other examples, the detectable protein isRuc-GFP and the therapeutic protein is anti-VEGF scAb (G6)-FLAG fusionprotein (e.g., GLV-1h107, GLV-1h109 and GLV-1h109).

Viruses that express both a detectable protein and therapeutic proteincan be used to detect and treat tumors. Such viruses can also beemployed to monitor tumor growth/regression over the course oftreatment, to monitor the efficacy of a particular tumor treatmentregimen or to monitor the efficacy of combinations of tumor treatments.The viruses can be modified express two or more therapeutic proteins toassess the efficacy of a combination of therapies.

C. METHODS FOR MODULATING VIRUS ATTENUATION

Provided herein are viruses, and methods for making and using suchviruses for therapeutic and diagnostic use. The methods provided hereininclude modulating the level of attenuation of a virus. The methods andexamples provided herein illustrate that attenuation can be modified byincreasing or decreasing the transcriptional and/or translational loadon the virus. For example, increasing the number of genes that the virusexpresses can cause competition for viral transcription and/ortranslation factors, which can result in changes in expression ofendogenous viral genes. Such changes can affect viral processes involvedin viral replication, thus contributing to the attenuation of the virus.For example, viral processes, such as viral DNA replication,transcription of other viral genes, viral mRNA production, viral proteinsynthesis, or virus particle assembly and maturation, can be affected.Insertion of gene expression cassettes that require binding of hostfactors for efficient transcription can be used to compete thetranscription and/or translation factors away from the endogenous viralpromoters and transcripts. For example, insertion of gene expressioncassettes that contain vaccinia strong late promoters into vacciniavirus can be used to attenuate expression of endogenous vaccinia lategenes.

Previous methods of altering attenuation of a virus that do noteliminate viral gene expression have relied on modifications of thenative viral gene promoter or enhancer regions to decrease expression ofa particular viral gene. In adenovirus, for example, decreasing the geneexpression of a selected gene region, the E4 transcription unit, hasbeen shown to decrease viral replication (Fang et al. (1997) J. Virol.71(6):4798-4803). Replacement of the promoter for the E4 transcriptionunit, which encodes several different proteins involved in DNAreplication, late-gene expression, and host gene shutoff, with asynthetic GAL4-responsive promoter led to an attenuation of the virus.Other studies in simian immunodeficiency virus (SIV) have shown thatexchange of the promoter enhancer region with that of cytomegalovirusimmediate early promoter (CMV-IE) resulted in an attenuated virus(Blancou et al. (2004) J. Virol. 78(3) 1080-1092). Modifying theattenuation in this system, however, is limited, since the options forreplacement of the promoter region are reduced to a single promoter orpromoters that maintain the proper timing of expression of the viralgene. Inducible promoters can be employed for gene expression; however,depending on the inducible system chosen, the expression of theessential gene(s) in vivo requires administration of the inducing agentto the host, which could be toxic. Furthermore, in order to achievegreater levels of attenuation of the virus, additional genes need to beselected for modification either by mutation or promoter replacement.Hence, adjusting the level of attenuation in such a system is difficultwithout modifying additional viral genes. Selection of propercombination of genes for modification is time-consuming and requiresextensive experimentation. For example, selection of additional genesfor promoter replacement requires that the decreased expression of aparticular gene is known to affect attenuation of the virus. In theabsence of experimentation, the attenuating effect of decreasedexpression is difficult to predict.

Provided herein are methods to attenuate a virus without the need toselect for individual viral genes to be modified. One of the advantagesto the methods provided herein is that attenuation of the virus does notrequire selecting various combinations of viral genes to be tested inorder to achieve a desired level of attenuation. Instead, the methodsprovide a predictable and systematic way of attenuating a virus bygenerating incremental decreases in viral gene expression by insertionof one or more gene expression cassettes that increase thetranscriptional and/or translational load on the virus. Therefore, themethods provide a way to alter viral gene expression without the need toselect specific viral genes for modification.

The methods provided herein can be used to increase or decrease theattenuation of a virus. In some embodiments, it can be desirable togenerate a more attenuated virus. A more attenuated virus can be lesstoxic to the host or be more suitable for particular routes ofadministration (e.g., systemic versus intratumoral). In otherembodiments, it can be desirable to generate a less attenuated virus. Aless attenuated virus can be more therapeutically effective (e.g., causemore tumor cell death) or be more suitable for particular routes ofadministration.

Any virus can be modified in accordance with the methods provided hereinto modulate attenuation, including but not limited to poxviruses,herpesviruses, adenoviruses, adeno-associated viruses, lentiviruses,retroviruses, rhabdoviruses, papillomaviruses, vesicular stomatitisvirus, measles virus, Newcastle disease virus, picornavirus, sindbisvirus, papillomavirus, parvovirus s, reovirus, coxsackievirus, influenzavirus, mumps virus, poliovirus, and semliki forest virus. In aparticular embodiment, the virus that is modified is a vaccinia virus.Methods for the generation of recombinant viruses using recombinant DNAtechniques are well known in the art (e.g., see U.S. Pat. Nos.4,769,330, 4,603,112, 4,722,848, 4,215,051, 5,110,587, 5,174,993,5,922,576, 6,319,703, 5,719,054, 6,429,001, 6,589,531, 6,573,090,6,800,288, 7,045,313, He et al. (1998) PNAS USA. 95(5): 2509-2514.Racaniello et al., (1981) Science 214: 916-919). Methods for thegeneration of recombinant vaccinia viruses for the methods can also befound in the Examples provided herein.

The methods provided herein include selection of attenuated viruses fortherapy and diagnosis. Exemplary uses for the viruses provided herein orgenerated by the methods provided herein include, but are not limited totherapy and diagnosis of conditions, such as neoplastic disease andother proliferative disorders and inflammatory disorders. The virusmediated treatment methods provided herein include administration ofviruses to hosts and accumulation of the viruses in the targeted cell ortissue, such as in a tumor which can result in lysing of the tumor cellsor leaking of tumor antigens, whereby an immune response againstreleased or leaked antigens is mounted. As a result, the tissues orcells in which the virus accumulates are inhibited.

In addition to the gene therapeutic methods of cancer treatment, liveattenuated viruses can be used for vaccination, such as in cancervaccination or antitumor immunity. Immunization, for example, against atumor can include a tumor-specific T-cell-mediated response throughvirally-delivered antigens or cytokines. To do so, the viruses can bespecifically targeted to the tumor tissues, with minimal infection toany other key organs and also can be modified or provided to produce theantigens and/or cytokines.

1. Expression Cassettes for Modulation of Attenuation

Provided herein are methods for modulating the level of attenuation of avirus by increasing or decreasing the transcriptional and/ortranslational load on the virus. According to the methods provided,attenuation can be modified by insertion, removal and/or modification ofheterologous DNA molecules in the viral genome. The methods providedherein for modulating the level of attenuation include insertion of oneor more heterologous gene expression cassettes, deletion of one or moregene expression cassettes, or modification one or more existing geneexpression cassettes.

a. Characteristics of an Expression Cassette

The heterologous DNA molecules for use in the methods provided aregenerally in the form of gene expression cassettes that contain apromoter operably linked to an open reading frame. For the methodsprovided herein, the open reading frame typically encodes anon-therapeutic gene, such as detectable protein, a protein capable ofproducing a detectable signal or other protein that does not produce atherapeutic effect. Exemplary non-therapeutic proteins include, but arenot limited to, proteins such as luciferases, fluorescent, or otherdetectable proteins as described elsewhere herein.

Although the methods provided herein for attenuation of a virustypically use an expression cassette that encodes a non-therapeuticprotein, the use of a gene expression cassette that encodes atherapeutic protein is not excluded. For example, an expression cassettethat encodes a therapeutic protein can be employed to enhance theattenuating effects of the expression cassette or to provide atherapeutic effect for treatment of a disease or condition.

In some embodiments, the expression cassette is transcribed by thevirus, but is not translated. Such cassettes can be employed to providetranscriptional load on the virus. For example, expression cassettes cancontain genes that do not encode a polypeptide (e.g., tRNA, rRNA,siRNA). In other examples, expression cassettes contain genes that arein the opposite orientation of the promoter. Hence, expression of suchcassettes produces an RNA transcript that is not translated.Alternatively, the expression cassette does not produce a transcript butstill can attenuate viral transcription by binding to viraltranscription factors.

In some alternative embodiments the expression cassette can contain anopen reading frame but lack a promoter. For example, an expressioncassette containing an open reading frame can be inserted into the viralgenome and be transcribed from an endogenous viral promoter ortranslated as part of a longer polypeptide. Exemplary of such insertionsare insertions into positive strand RNA viral genomes where severalgenes are translated into a single polypeptide. Insertion of an openreading frame in a positive strand RNA virus increases the number ofgenes that are translated and thus increases the length of thepolypeptide that is translated. As a result, there can be increased loadon the viral translation machinery, leading to an attenuation of thevirus. In another example, an open reading frame can be inserted into anegative strand RNA virus, such as vesicular stomatitis virus (VSV),which transcribes long mRNA transcripts encoding several genes.Insertion of an open reading frame in a negative strand RNA virusincreases the number of genes that are transcribed and thus increasesthe length of the transcript and the number of genes that can betranslated. As a result there can be increased load on the viraltranscription and translation machinery. Positioning of an inserted openreading frame within a VSV genome also has been shown to affect thelevel of attenuation of the virus (see e.g., U.S. Pat. No. 6,777,220).One or more open reading frames can thus be positioned within the viralgenome to generate the desired level of attenuation.

i. Expression Cassette Promoters

Competition of promoters for transcription factors as a mechanism ofcontrolling gene expression that has been observed in studies of bothendogenous and transient eukaryotic transcription and viraltranscription (see e.g., Raju et al. (1991) J. Virol. 65(5) 2501-2510;Latchman et al. (1989) Nucl. Acids Res. 17(21) 8533-8541; Hsue andMasters (1999) J. Virol. 73(7): 6128-6135; Foley et al. (1992) Genes &Dev. 6:730-744; Keegan et al., (1986) Science 231:699-704). For themethods provided herein, promoter competition that involves competitionfor transcription factors can be for either viral or host proteins thatcontribute to viral replication and production of viral particles.

Exemplary promoters useful in the methods provided herein include anypromoter useful for driving the expression of the open reading frame ofthe expression cassette. The promoters can be any eukaryotic or viralpromoter that functions within the cell to be infected. Choice of apromoter for the use in an expression cassette depends on a variety offactors, including, for example, the characteristics of the virus (i.e.,cytoplasmic or nuclear, RNA or DNA, segmented or non-segmented, positivesense or negative sense genome), the timing of expression desired, andthe strength of expression desired. For example, vaccinia viruses, whichare cytoplasmic DNA viruses, employ promoters that are active in thecytoplasm of the infected cell. Such promoters typically use mostlyviral factors for DNA replication and transcription, though some hostfactors, such as YY1 and TBP, are transported from the nucleus to thecytoplasm to aid in viral replication (see e.g., Davison and Moss (1989)J. Mol. Biol. 210: 749-769; Davison and Moss (1989) J. Mol. Biol. 210:771-784; Weir and Moss (1987) J. Virol. 61(1): 75-80; Broyles et al.(1999) J. Biol. Chem. 274(50) 35662-35667; Broyles (2003) J. Gen. Virol.84:2293-2903; Knutson et al. (2006) J. Virol. 80 (14) 6784-6793). Hence,promoters for use in an expression cassette for modulating theattenuation of a vaccinia virus include, but are not limited to,vaccinia and other poxviruses' (e.g. cowpox, ectromelia, monkeypox,camelpox, variola, canarypox, fowlpox, myxoma, rabbit fibroma, goatpox,sheeppox, sealpox, swinepox, tanapox, yaba monkey tumor, and molluscumcontagiosum) natural promoters and synthetic promoters. Exemplaryvaccinia natural promoters include, but are not limited to, 7.5K, H5R,TK, P28, P11k, C11R, G8R, F17R, 13L, 18R, A1L, A2L, A3L, H1L, H3L, H5L,H6R, H8R, D1R, D4R, D5R, D9R, D11L, D12L, D13L, M1L, N2L, P4b or K1L.Another non-limiting exemplary poxvirus natural promoter includes cowpoxATI (A-type inclusion body). Exemplary vaccinia synthetic promotersinclude, but are not limited to, a synthetic early promoter (P_(SE)), asynthetic late promoter (P_(SL)), a synthetic early/late promoter(P_(SEL)) and chimeric promoters between early and late poxyiralpromoters (see e.g., Chakrabarti et al. (1997) BioTechniques 23(6)1094-1097; Patel et al., (1988) Proc. Natl. Acad. Sci. USA 85,9431-9435; Davison and Moss (1990) Nuc. Acids Res. 18(14): 4285-4286;Hammond et al. (1997) J. Virol. Meth. 66: 135-138; Kumar and Boyle(1990) Virology 179: 151-158). Davison and Moss (1989) J. Mol. Biol.210: 749-769; Davison and Moss (1989) J. Mol. Biol. 210: 771-784;Baldick et al. (1992) J. Virol. 66: 4710-4719.).

In some embodiments, it can be desirable to choose promoters providedexpressed at a particular time points in the viral life cycle. Forexample, promoters useful in the methods provided herein for modulatingthe attenuation of a virus, such as a vaccinia virus, can be an earlypromoter, an intermediate promoter, or late promoter. In otherembodiments it can be useful to use a promoter that is active throughoutthe viral life cycle. Exemplary promoters in vaccinia virus that areexpressed throughout the life cycle include tandem arrangements ofvaccinia early and intermediate or late promoters (see e.g. Wittek etal. (1980) Cell 21: 487-493; Broyles and Moss (1986) Proc. Natl. Acad.Sci. USA 83: 3141-3145; Ahn et al. (1990) Mol. Cell. Biol. 10:5433-54441; Broyles and Pennington (1990) J. Virol. 64: 5376-5382). Anexemplary vaccinia early promoter is a synthetic early promoter(P_(SE)), which typically initiate gene expression from 0-3 hours postinfection. Exemplary vaccinia late promoters include, but are notlimited to, a vaccinia 11k promoter (P_(11k)) and a synthetic latepromoter (P_(SL)), which typically initiate gene expression 2-3 hourspost-infection. Exemplary vaccinia early/late promoters that expressthroughout the vaccinia life cycle include, but are not limited to, a7.5K promoter (P_(7.5k)) and a synthetic early/late promoter (P_(SEL)).

In some embodiments, it can be desirable to choose a promoter of aparticular relative strength. Hence, promoter potency can be used toinfluence the degree of competition between the inserted heterologousexpression cassettes and endogenous genes. For example, in vaccinia,synthetic early/late P_(SEL) and many late promoters (e.g., P_(11k) andP_(SL)) are relatively strong promoters, whereas vaccinia syntheticearly, P_(SE), P_(7.5k) early/late, P_(7.5k) early, and P₂₈ latepromoters are relatively weaker promoters (see e.g., Chakrabarti et al.(1997) BioTechniques 23(6) 1094-1097). In some embodiments, a strongerpromoter is employed in an expression cassette to provide bettercompetition for viral factors and thus generate a more attenuated virus.In other embodiments, a weaker promoter is employed in an expressioncassette to provide a less attenuating effect. In some embodiments,where more than one heterologous expression cassette is inserted into aviral genome, combinations of promoters that differ in strength can beemployed to fine tune attenuation of the virus. For example, if two ormore expression cassettes are inserted into a viral genome, some of theexpression cassettes can contain a strong viral promoter while otherexpression cassettes can contain a weaker promoter.

ii. Insertion Sites for Expression Cassettes

Sites for the insertion of heterologous nucleic acid molecules are knownin the art and have been described for various viral vectors (see e.g.,U.S. Pat. Nos. 5,166,057, 5,266,489, 6,338,846, 6,248,320, 6,221,646,6,841,158, 7,101,685, 7,001,760 and references therein). Heterologousnucleic acid molecules are typically inserted into a non-coding regionor in a coding region for a gene that is nonessential for viralreplication. For example, in vaccinia virus, sites for insertions ofheterologous DNA molecules can be in intergenic regions, non-codingregions, and or nonessential genes or gene regions including, but notlimited to, thymidine kinase (TK) gene, hemagglutinin (HA) gene, F14.5L(see, e.g., U.S. Patent Pub. No. 2005-0031643), VGF gene (see, e.g.,U.S. Pat. Pub. No. 2003-0031681), Hind III F, F13L, or Hind III M (see,e.g., U.S. Pat. No. 6,548,068); a hemorrhagic region or an A typeinclusion body region (ATI) (see, e.g., U.S. Pat. Nos. 6,265,189 and6,596,279); A33R, A34R, A36R or B5R genes (see, e.g., Katz et al.,(2003) J. Virology 77:12266-12275); SalF7L (see, e.g., Moore et al.,(1992) EMBO J. 11:1973-1980); NIL (see, e.g., Kotwal et al. (1989)Virology 171:579-587); M1 lambda (see, e.g., Child et al. (1990)Virology. 174:625-629); HR, HindIII-MK, HindIII-MKF, HindIII-CNM, RR, orBamF (see, e.g., Lee et al. (1992) J. Virol. 66:2617-2630); C21L (see,e.g., Isaacs et al. (1992) Proc Natl Acad Sci USA. 89:628-632), hostrange region genes K1L and C7L, A35R (see e.g., U.S. Pat. Nos.6,265,189, 7,045,313; U.S. Patent Pub. Nos. 2005-0244428, 2006-0159706;Coupar et al. J. Gen. Virol. (2000) 81: 431-439; Smith et al. (1993)Vaccine 11(1): 43-53). If more than one gene expression cassette isinserted, the insertions can be at the same insertion site or differentinsertion sites. Alternatively, the heterologous nucleic acid moleculescan be inserted into an essential gene, and a cell line for packaging ofthe virus could be use for the production of the virus.

Mutation of nonessential vaccinia genes can also contribute to increasedattenuation of the virus. Thus, insertion of heterologous expressioncassettes into a nonessential gene, such as the TK gene, can attenuatethe virus in two aspects: by gene mutation and by added transcriptionaland/or translational load. For the methods provided herein, mutation ofnonessential genes is not required; however, one or more nonessentialgene can be modified to enhance the attenuating effects of the geneexpression cassette. The attenuation of the virus can be subsequentlylessened (i.e., the virus exhibits increased replication) by removingthe expression cassette and replacing it with noncoding sequence so thatthe gene remains inactive. Thus, removal or replacement of a geneexpression cassette decreases the transcriptional and/or translationalload on the virus, resulting in a decrease in attenuation of the virus.As supported in the Examples, replacement of a gene expression cassettefrom the HA gene, the TK gene, the F14.5L locus, or a combinationthereof, can lead to increased replication of the virus both in vitroand in vivo.

b. Insertion or Removal of Expression Cassettes

An increase in the number of gene expression cassettes in the viralgenome can lead to a competition for the transcriptional and/ortranslational machinery of the virus since the factors needed totranscribe and translate the gene expression cassette are also needed bythe virus for expression of endogenous viral genes. Hence, increasingthe number of heterologous expression cassettes in the viral genome canincrease the transcriptional and translational load on the virus. As aresult, expression of viral proteins needed in the production new viralparticles is decreased, thus generating a more attenuated virus.Exemplary mechanisms of competition include, but are not limited to,inhibition of transcription initiation by binding a transcription factoror binding limiting amounts of polymerase, limiting nucleotide pools fortranscription, binding a termination factor, which can tie up thepolymerase, binding limiting amounts of ribosomes or translationfactors, and limiting tRNA pools and translation termination factors.

In some embodiments, a virus to be modified contains one or moreheterologous gene expression cassettes. According to the methodsprovided herein, removal of a gene expression cassette from such a viruscan decrease the transcriptional and/or translational load on the virus.This can result in less competition for expression viral genes, and thusproducing a less attenuated virus. In some embodiments, the geneexpression cassette to be removed occurs within a viral gene. Typically,removal of the gene expression cassette does not restore the function ofthe viral gene. In a particular embodiment, the gene expression cassetteis removed from the viral genome and replaced with a non-codingsequence.

Insertion and removal of expression cassettes can be carried out usingany known method in the art for modification of a viral genome. Invaccinia, for example, well-known techniques for insertion or removal ofheterologous nucleic acid molecules by homologous recombination areavailable. Typically, the methods involve generating a shuttle plasmidvector, containing the expression cassette flanked by vaccinia virusDNA. The shuttle vector is then transfected into cells that have beeninfection with the target vaccinia virus. Homologous recombinationoccurs between the shuttle vector and the vaccinia virus genome at a lowfrequency to generate the modified virus with the inserted expressioncassette. Shuttle vectors for use in the methods provided herein formodification of a vaccinia virus or for construction of new shuttlevectors for modification of a vaccinia virus include any known shuttlevector in the art including, but not limited to, pGS20, pSC59, pMJ601,pSC65 (SEQ ID NO: 30), pSC11, pMCO2, pCF11, PTKgptF1s, pMC1107, pNCVVhaT(SEQ ID NO: 4), pNCVVf14.51T (SEQ ID NO: 11), pCR-TKLR-gpt2 (SEQ ID NO:17), and other newly made vectors provided herein among others.Exemplary shuttle vectors for use in the methods herein are alsoprovided in the Examples.

c. Modification of Expression Cassettes

In some embodiments, where a virus to be modified contains one or moreheterologous gene expression cassettes, a gene expression cassette canbe modified to increase or decrease the transcriptional and/ortranslational competition for expression of viral genes. Expressioncassettes can be modified, for example, by modification of the promoteror other non-coding portion of the expression cassette or bymodification of the open reading frame of the expression cassette.

i. Promoter Modification

Promoters of expression cassettes can be modified by exchanging thepromoter for a different promoter to alter the level of attenuationcaused by the expression cassette. Modifications of an expressioncassette in a virus include exchanging the promoter region of theexpression cassette or exchanging the gene expression cassette withanother gene expression cassette with a different promoter. The changein promoter can, for example, alter the level of transcription and/ortranslation of the expression cassette or alter the timing of expressionof the gene(s) encoded by the expression cassette. Exemplary changes ina promoter include increasing or decreasing the strength of the promoteror exchanging the promoter such that the gene(s) encoded by the openreading frame is expressed at different time in the viral life cycle.Stronger promoters in an expression cassette typically can providebetter competition for viral factors and thus generate a more attenuatedvirus, whereas weaker promoters provide lesser amount of competitioncompared to stronger promoters and thus generate a lesser degree ofattenuation.

ii. Modification of Open Reading Frame

Expression cassettes can also be modified, for example, by altering theopen reading frame (ORF) of the expression cassette. The ORF can bemodified to modulate the level of attenuation generated by theexpression cassette. The ORF of the expression cassette can be exchangedfor another ORF or a modified ORF, the entire gene expression cassettecan also be exchanged with another cassette with a different ORF or amodified ORF. Exemplary modifications to an ORF include, but are notlimited to, altering the length of the ORF, the genes encoded by theORF, or the number of genes encoded by the ORF. The length of the ORFcan be modified such that a longer mRNA is transcribed or a longerpolypeptide is translated. For example, the ORF can be modified byincreasing the length of the existing coding sequence or substitutingthe ORF for a longer ORF. RNA viruses, such as VSV and New CastleDisease Virus (NDV), for example, are particularly sensitive to lengthof inserted ORFs since transcription of the viral genome involvesproduction of long mRNA transcripts encoding multiple genes (see e.g.,U.S. Pat. No. 6,713,066, Barr et al. (2002) Biochim. et Biophys. Acta1577:337-353; Krishnamurthy et al. (2000) Virology 278(1):168-182).

The ORF can also be modified to generate more than one polypeptide. ORFsthat encode more than one polypeptide can generate a fusion protein ofthe two polypeptides or can include internal ribosome binding sites(IRES) that separate the coding sequences for production of the two ormore separate polypeptides. Alternatively, the ORF can include anintervening coding sequence that allows cleavage of the two polypeptidespost-translationally. For example, the picornavirus 2A element can beinserted in between and in frame with two coding sequences, such thatthe two polypeptides are proteolytically cleaved by the host or by avirally encoded factor (see e.g., Osborn et al. (2005) Mol. Ther.12(3):569-74). An exemplary virus provided herein that employs a geneexpression cassette containing the picornavirus 2A element separatingthe genes encoding click beetle luciferase and red fluorescent proteinis GLV-1h84.

ORFs can also be modified to reduce the level of attenuation. Forexample, an ORF of an expression cassette can be shortened, substitutedfor shorter ORF or deleted. Alternatively, an ORF can be exchanged for anon-coding sequence or a coding sequence that is in the reverseorientation to the promoter, resulting in removal of a translationalunit from the virus. Hence, the level of attenuation can be decreased bydecreasing the translational load caused by translation of the ORF ofthe expression cassette.

2. Transcription Factor Decoys

In one embodiment, the heterologous DNA molecule that can be insertedinto the viral genome contains one or more binding sites fortranscription factors needed for viral transcription, but is notoperably linked to an open reading frame. For example, the DNA sequencecan be a promoter sequence that is not operably linked to an openreading frame. The heterologous DNA can act as a decoy that can bind toviral transcription factors or host transcription factors involved inviral transcription. The use of decoys in the form of oligonucleotideshas been used successfully to inhibit eukaryotic and viral transcription(see e.g. U.S. Pat. Nos. 5,712,384, 5,683,985, and 6,821,956; U.S.Patent Pub. No. 2004-0127446; Michienzi et al. Proc. Natl. Acad. Sci.USA 99(22): 14047-14052; Tomita et al. (2004) Interntl. J. Mol. Med. 13:629-636; Cho-Chung et al. (2000) Mol. Cell. Biochem. 212(1-2): 29-34;Seki et al (2006) Mol. Caner. Ther. 5: 985-994). In the methods providedherein, insertion of heterologous DNA into a viral genome can lead to acompetition for the transcriptional machinery of the virus since thefactors that bind to the inserted sequence are needed by the virus forexpression of endogenous viral genes. Hence, increasing the numberheterologous binding sites for factors needed for viral transcriptioncan result in a decrease in production of viral proteins needed toproduce new viral particles, thus generating a more attenuated virus.

3. Fine Tuning Attenuation—Combinations of Insertions, Deletions, orModifications

The steps provided herein for modulating a virus to increase or decreasethe attenuation of the virus can be repeated to achieve a desired levelof attenuation. For example, additional modification of the virus can becarried out to improve the level of attenuation desired. The level ofattenuation can be assessed in vitro or in vivo, and a determination canbe made whether additional modification of the virus is preferred. Thevirus can then be modified by insertion, removal and/or modification oradditional expression cassettes or be further modified as discussedbelow.

Combinations of insertions, deletions or modifications of expressioncassettes can be carried out to achieve the desired level ofattenuation. In a particular embodiment, a heterologous nucleic acidmolecule is inserted, removed and/or modified in the viral genome. In afurther embodiment, two or more heterologous nucleic acid molecules areinserted, removed and/or modified in the viral genome. If two or moreheterologous nucleic acid molecules are inserted, removed and/ormodified in the viral genome, one or more nucleic acid molecules can beinserted while one or more nucleic acid molecules is removed. Similarly,one or more heterologous nucleic acid molecules can be inserted whileone or more heterologous nucleic acid molecules modified. Further, oneor more nucleic acid molecules can be removed while one or more nucleicacid molecules is modified. The heterologous nucleic acid molecules cancontain an open reading frame, lack an open reading frame, or be acombination of both.

4. Assays for Attenuated Viruses

Methods for assessing the level of attenuation of a virus by in vitroand in vivo methods are known in the art and include, but are notlimited to, methods such as plaque assays and mouse models of viralpathogenicity. Exemplary methods for studying vaccinia early,intermediate, and late transcription can be found in Broyles et al.Methods Mol. Biol. (2004) 269:135-142 and Wright et al. Methods Mol.Biol. (2004) 269:143-150. Method for assaying for viral RNA transcriptsand proteins include, but are not limited to, well-known techniques asRNA hybridization and blotting techniques and immunohistochemistry.

D. FURTHER MODIFICATIONS

Viruses provided herein and viruses produced by the methods providedherein can be further modified by any known method for modifying avirus. Furthermore, viruses provided herein and viruses produced by themethods provided herein can be further modified to attenuate the virus.Hence, the methods provided herein can be combined with any known methodfor modifying a virus. Furthermore, the methods provided herein can becombined with any known method for modulating the attenuation of avirus. For example, such methods include modification of one or moreviral genes, such as by a point mutation, a deletion mutation, aninterruption by an insertion, a substitution, or a mutation of the viralgene promoter or enhancer regions. Modifications in a viral gene can beintroduced into the virus prior to the execution of the methods ofmodulating attenuation provided herein. Alternatively, modifications canbe introduced into the virus following the execution of the methods ofmodulating attenuation provided herein. Modifications in a viral genecan be combined with the methods provided herein or used to modify theviruses provided to either increase or decrease the attenuation of thevirus. Further modifications of a virus that are combined with themethods provided herein or used to modify the viruses provided, however,are not required to affect the attenuation of the virus.

Further modifications of the viruses provided can enhance one or morecharacteristics of the virus. Such characteristics can include, but arenot limited to, attenuated pathogenicity, reduced toxicity, preferentialaccumulation in tumor, increased ability to activate an immune responseagainst tumor cells, increased immunogenicity, increased or decreasedreplication competence, and are able to express exogenous proteins, andcombinations thereof. In some embodiments, the modified viruses have anability to activate an immune response against tumor cells withoutaggressively killing the tumor cells. In other embodiments, the virusescan be modified to express one or more detectable genes, including genesthat can be used for imaging. In other embodiments, the viruses can bemodified to express one or more genes for harvesting the gene productsand/or for harvesting antibodies against the gene products.

Conventional methods for attenuation of a virus include mutation inviral virulence genes, such as by a point mutation, a deletion mutation,an interruption mutation, or modification of the virulence gene promoteror enhancer regions. The methods provided herein for increasing ordecreasing the attenuation of a virus typically do not requiremodification viral genes. Modification of viral genes, however can becombined with the methods provided to alter the level of attenuation.

1. Modification of Viral Genes

Methods for modifying a virus include modifications in one or more viralgenes. Modification can include those that inactivate viral gene orabolish or decrease the activity of a viral gene product. Suchmodifications in a viral gene can alter the viral processes, such as,for example, viral infectivity, viral DNA replication, viral proteinsynthesis, virus particle assembly and maturation, and viral particlerelease. Exemplary viral genes for modification include, but are notlimited to, viral surface antigens (e.g. proteins that mediate viralattachment to host cell receptors), viral proteases, and viral enzymesinvolved in viral replication and transcription of viral genes (e.g.,polymerases, replicases and helicases). Modifications in such genes candecrease the overall replication of the virus and production of viralparticles thus resulting in a more attenuated virus.

In another embodiment, a viral surface antigen gene can be modified toproduce a chimeric protein such that the heterologous epitope isexpressed on the surface of the virus. Viruses expressing such chimericproteins are thus useful as vaccines for use in generating an immuneresponse in the host subject. Exemplary epitopes include but are notlimited to tumor antigens, viral and bacterial antigens. Many exemplaryantigens are known in the art, and include, for example, those listedand/or described in Novellino et al. (2005) Cancer Immunol Immunother.54(3):187-207; Eisenberger et al. (2006) Hematol Oncol Clin North Am.20(3):661-87. In one embodiment, insertion of a heterologous epitopeinto the viral gene can affect the level of attenuation of the virus. Inan alternative embodiment, the level of attenuation of the virus isunaffected by insertion of a heterologous epitope into the viral gene.

2. Expression of Additional Heterologous Genes

Viruses provided herein and viruses generated using the methods providedherein can be further modified to express one or more additionalheterologous genes. Gene expression can include expression of a proteinencoded by a gene and/or expression of an RNA molecule encoded by agene. In some embodiments, the viruses can express heterologous genes atlevels high enough that permit harvesting products of the heterologousgene from the tumor.

Expression of heterologous genes can be controlled by a constitutivepromoter, or by an inducible promoter. Expression can also be influencedby one or more proteins or RNA molecules expressed by the virus. Anexemplary inducible promoter system can include a chimeric transcriptionfactor comprising a yeast GAL4 DNA-binding domain fused to a ligandbinding domain derived from a progesterone receptor and to theactivation domain of the herpes simplex virus protein VP16, and asynthetic promoter containing a series of GAL4 recognition sequencesupstream of the adenovirus major late E1B TATA box, linked to one ormore heterologous genes; in this exemplary system, administration ofmifepristone (RU486) to a subject can result in induction of theheterologous genes. Other exemplary inducible promoter systems include,but are not limited to, a tetracycline-repressed regulated system,ecdysone-regulated system, and rapamycin-regulated system(Agha-Mohammadi and Lotze (2000) J. Clin. Invest. 105(9): 1177-1183).Heterologous genes expressed can include genes encoding a therapeuticgene product, genes encoding a detectable gene product, such as a geneproduct that can be used for imaging, genes encoding a gene product tobe harvested, genes encoding an antigen of an antibody to be harvestedor to elicit an immune response. The viruses provided herein can be usedfor expressing genes in vivo and in vitro. Exemplary proteins includereporter proteins (E. coli β-galactosidase, β-glucuronidase,xanthineguanine phosphoribosyltransferase), proteins facilitatingdetection, such as a detectable protein or a protein capable of inducinga detectable signal, (luciferase, fluorescent proteins, transferrinreceptor, for example), proteins useful for tumor therapy (pseudomonas Aendotoxin, diphtheria toxin, p53, Arf, Bax, tumor necrosis factor-alpha,HSV TK, E. coli purine nucleoside phosphorylase, angiostatin,endostatin, cytokines, or chemokines) and other proteins.

a. Detectable Gene Product

Viruses provided herein and viruses generated using the methods providedherein can express one or more genes whose products are detectable orwhose products can provide a detectable signal. A variety of detectablegene products, such as detectable proteins are known in the art, and canbe used with the viruses provided herein. Detectable proteins includereceptors or other proteins that can specifically bind a detectablecompound, proteins that can emit a detectable signal such as afluorescence signal, and enzymes that can catalyze a detectable reactionor catalyze formation of a detectable product.

In some embodiments, the virus expresses a gene encoding a protein thatcan emit a detectable signal or that can catalyze a detectable reaction.A variety of DNA sequences encoding proteins that can emit a detectablesignal or that can catalyze a detectable reaction, such as luminescentor fluorescent proteins, are known and can be used in the viruses andmethods provided herein. Exemplary genes encoding light-emittingproteins include genes from bacterial luciferase from Vibrio harveyi(Belas et al., Science 218 (1982), 791-793), bacterial luciferase fromVibrio fischerii (Foran and Brown, Nucleic acids Res. 16 (1988), 177),firefly luciferase (de Wet et al., Mol. Cell. Biol. 7 (1987), 725-737),aequorin from Aequorea victoria (Prasher et al., Biochem. 26 (1987),1326-1332), Renilla luciferase from Renilla renformis (Lorenz et al,PNAS USA 88 (1991), 4438-4442) and green fluorescent protein fromAequorea victoria (Prasher et al., Gene 111: 229-233 (1987)). The luxAand luxB genes of bacterial luciferase can be fused to produce thefusion gene (Fab₂), which can be expressed to produce a fully functionalluciferase protein (Escher et al., PNAS 86: 6528-6532 (1989)).Transformation and expression of these genes in viruses can permitdetection of viral infection, for example, using a low light and/orfluorescence imaging camera. In some embodiments, luciferases expressedby viruses can require exogenously added substrates such as decanal orcoelenterazine for light emission. In other embodiments, viruses canexpress a complete lux operon, which can include proteins that canprovide luciferase substrates such as decanal. For example, virusescontaining the complete lux operon sequence, when injectedintraperitoneally, intramuscularly, or intravenously, allowed thevisualization and localization of microorganisms in live mice indicatingthat the luciferase light emission can penetrate the tissues and can bedetected externally (Contag et al. (1995) Mol. Microbiol. 18: 593-603).

In other embodiments, the virus can express a gene that can bind adetectable compound or that can form a product that can bind adetectable compound. A variety of gene products, such as proteins, thatcan specifically bind a detectable compound are known in the art,including receptors, metal binding proteins (e.g., siderophores,ferritins, transferrin receptors), ligand binding proteins, andantibodies. Any of a variety of detectable compounds can be used, andcan be imaged by any of a variety of known imaging methods. Exemplarycompounds include receptor ligands and antigens for antibodies. Theligand can be labeled according to the imaging method to be used.Exemplary imaging methods include any of X-rays, a variety magneticresonance methods such as magnetic resonance imaging (MRI) and magneticresonance spectroscopy (MRS), and also include any of a variety oftomographic methods including computed tomography (CT), computed axialtomography (CAT), electron beam computed tomography (EBCT), highresolution computed tomography (HRCT), hypocycloidal tomography,positron emission tomography (PET), single-photon emission computedtomography (SPECT), spiral computed tomography and ultrasonictomography.

Labels appropriate for X-ray imaging are known in the art, and include,for example, Bismuth (III), Gold (III), Lanthanum (III) or Lead (II); aradioactive ion, such as ⁶⁷Copper, ⁶⁷Gallium, ⁶⁸Gallium, ¹¹¹Indium,¹¹³Indium, ¹²³Iodine, ¹²⁵Iodine, ¹³¹Iodine, ¹⁹⁷Mercury, ²⁰³Mercury,¹⁸⁶Rhenium, ¹⁸⁸Rhenium, ⁹⁷Rubidium, ¹⁰³Rubidium, ⁹⁹Technetium or⁹⁰Yttrium; a nuclear magnetic spin-resonance isotope, such as Cobalt(II), Copper (II), Chromium (III), Dysprosium (III), Erbium (III),Gadolinium (III), Holmium (III), Iron (II), Iron (III), Manganese (II),Neodymium (III), Nickel (II), Samarium (III), Terbium (III), Vanadium(II) or Ytterbium (III); or rhodamine or fluorescein.

Labels appropriate for magnetic resonance imaging are known in the art,and include, for example, gadolinium chelates and iron oxides. Use ofchelates in contrast agents is known in the art. Labels appropriate fortomographic imaging methods are known in the art, and include, forexample, β-emitters such as ¹¹C, ¹³N, ¹⁵O or ⁶⁴Cu or (b) γ-emitters suchas ¹²³I. Other exemplary radionuclides that can, be used, for example,as tracers for PET include ⁵⁵Co, ⁶⁷Ga, ⁶⁸Ga, ⁶⁰Cu(II), ⁶⁷Cu(II), ⁵⁷Ni,⁵²Fe and ¹⁸F (e.g., ¹⁸F-fluorodeoxyglucose (FDG)). Examples of usefulradionuclide-labeled agents are ⁶⁴Cu-labeled engineered antibodyfragment (Wu et al. (2002) PNAS USA 97: 8495-8500), ⁶⁴Cu-labeledsomatostatin (Lewis et al. (1999) J. Med. Chem. 42: 1341-1347),⁶⁴Cu-pyruvaldehyde-bis(N4methylthiosemicarbazone) (64Cu-PTSM) (Adonai etal. (2002) PNAS USA 99: 3030-3035), ⁵²Fe-citrate (Leenders et al. (1994)J. Neural. Transm. Suppl. 43: 123-132), ⁵²Fe/^(52m)Mn-citrate (Calonderet al. (1999) J. Neurochem. 73” 2047-2055) and ⁵²Fe-labeled iron (III)hydroxide-sucrose complex (Beshara et al. (1999) Br. J. Haematol. 104:288-295, 296-302).

In some examples dual imaging in vitro and/or in vivo can be used todetect two or more detectable gene products, gene products that producea detectable signal, gene products that can bind a detectable compound,or gene products that can bind other molecules to form a detectableproduct. In some examples, the two or more gene products are expressedby different viruses, whereas in other examples the two or more geneproducts are produced by the same virus. For example, a virus canexpress a gene product that emits a detectable signal and also express agene product that catalyzes a detectable reaction. In other examples, avirus can express one or more gene products that emit a detectablesignal, one or more gene products that catalyze a detectable reaction,one or more gene products that can bind a detectable compound or thatcan form a detectable product, or any combination thereof. Anycombination of such gene products can be expressed by the virusesprovided herein and can be used in combination with any of the methodsprovided herein. Imaging of such gene products can be performed, forexample, by various imaging methods as described herein and known in theart (e.g., fluorescence imaging, MRI, PET, among may other methods ofdetection). Imaging of gene products can also be performed using thesame method, whereby gene products are distinguished by theirproperties, such as by differences in wavelengths of light emitted. Forexample, a virus can express more than one fluorescent protein thatdiffers in the wavelength of light emitted (e.g., a GFP and an RFP). Inanother non-limiting example, an RFP can be expressed with a luciferase.In yet other non-limiting examples, a fluorescent gene product can beexpressed with a gene product, such as a ferritin or a transferrinreceptor, used for magnetic resonance imaging. A virus expressing two ormore detectable gene products or two or more viruses expressing two ormore detectable gene products can be imaged in vitro or in vivo usingsuch methods. In some embodiments the two or more gene products areexpressed as a single polypeptide, such as a fusion protein. For examplea fluorescent protein can be expressed as a fusion protein with aluciferase protein.

b. Therapeutic Gene Product

Viruses provided herein and viruses generated using the methods providedherein can express one or more genes whose products cause cell death orwhose products cause an anti-tumor immune response; such genes can beconsidered therapeutic genes. A variety of therapeutic gene products,such as toxic or apoptotic proteins, or siRNA, are known in the art, andcan be used with the viruses provided herein. The therapeutic genes canact by directly killing the host cell, for example, as a channel-formingor other lytic protein, or by triggering apoptosis, or by inhibitingessential cellular processes, or by triggering an immune responseagainst the cell, or by interacting with a compound that has a similareffect, for example, by converting a less active compound to a cytotoxiccompound. A large number of therapeutic proteins that can be expressedfor tumor treatment are known in the art, including, but not limited to,tumor suppressors, toxins, cytostatic proteins, and costimulatorymolecules such as cytokines and chemokines. Costimulatory molecules forthe methods provided herein include any molecules which are capable ofenhancing immune responses to an antigen/pathogen in vivo and/or invitro. Costimulatory molecules also encompass any molecules whichpromote the activation, proliferation, differentiation, maturation, ormaintenance of lymphocytes and/or other cells whose function isimportant or essential for immune responses. An exemplary, non-limitinglist of therapeutic proteins includes WT1, p53, p16, Rb, BRCA1, cysticfibrosis transmembrane regulator (CFTR), Factor VIII, low densitylipoprotein receptor, beta-galactosidase, alpha-galactosidase,beta-glucocerebrosidase, insulin, parathyroid hormone,alpha-1-antitrypsin, rsCD40L, Fas-ligand, TRAIL, TNF, antibodies,microcin E492, diphtheria toxin, Pseudomonas exotoxin, Escherichia coliShiga toxin, Escherichia coli Verotoxin 1, and hyperforin. Exemplarycytokines include, but are not limited to, chemokines and classicalcytokines, such as the interleukins, including for example,interleukin-1, interleukin-2, interleukin-6 and interleukin-12, tumornecrosis factors, such as tumor necrosis factor alpha (TNF-α),interferons such as interferon gamma (IFN-γ), granulocyte macrophagecolony stimulating factor (GM-CSF) and exemplary chemokines including,but not limited to CXC chemokines such as IL-8 GROα, GROβ, GROγ, ENA-78,LDGF-PBP, GCP-2, PF4, Mig, IP-10, SDF-1α/β, BUNZO/STRC33, I-TAC,BLC/BCA-1; CC chemokines such as MIP-1α, MIP-1β, MDC, TECK, TARC,RANTES, HCC-1, HCC-4, DC-CK1, MIP-3α, MIP-3β, MCP-1, MCP-2, MCP-3,MCP-4, Eotaxin, Eotaxin-2/MPIF-2, 1-309, MIP-5/HCC-2, MPIF-1, 6Ckine,CTACK, MEC; lymphotactin; and fractalkine. Exemplary other costimulatorymolecules include immunoglobulin superfamily of cytokines, such as B7.1,B7.2.

In other embodiments, the viruses can express a protein that converts aless active compound into a compound that causes tumor cell death.Exemplary methods of conversion of such a prodrug compound includeenzymatic conversion and photolytic conversion. A large variety ofprotein/compound pairs are known in the art, and include, but are notlimited to, Herpes simplex virus thymidine kinase/ganciclovir, Herpessimplex virus thymidine kinase/(E)-5-(2-bromovinyl)-2′-deoxyuridine(BVDU), varicella zoster thymidine kinase/ganciclovir, varicella zosterthymidine kinase/BVDU, varicella zoster thymidinekinase/(E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU),cytosine deaminase/5-fluorouracil, cytosine deaminase/5-fluorocytosine,purine nucleoside phosphorylase/6-methylpurine deoxyriboside, betalactamase/cephalosporin-doxorubicin, carboxypeptidaseG2/4-[(2-chloroethyl) (2-mesuloxyethyl)amino]benzoyl-L-glutamic acid(CMDA), carboxypeptidase A/methotrexate-phenylamine, cytochromeP450/acetominophen, cytochrome P450-2B1/cyclophosphamide, cytochromeP450-4B1/2-aminoanthracene, 4-ipomeanol, horseradishperoxidase/indole-3-acetic acid, nitroreductase/CB1954, rabbitcarboxylesterase/7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin(CPT-11), mushroomtyrosinase/bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28,beta galactosidase/1-chloromethyl-5-hydroxy-1,2-dihyro-3H-benz[e]indole,beta glucuronidase/epirubicin glucuronide, thymidinephosphorylase/5′-deoxy-5-fluorouridine, deoxycytidine kinase/cytosinearabinoside, and linamerase/linamarin.

In another embodiment, the therapeutic gene product can be an siRNAmolecule. The siRNA molecule can be directed against expression of atumor-promoting gene, such as, but not limited to, an oncogene, growthfactor, angiogenesis promoting gene, or a receptor. The siRNA moleculealso can be directed against expression of any gene essential for cellgrowth, cell replication or cell survival. The siRNA molecule also canbe directed against expression of any gene that stabilizes the cellmembrane or otherwise limits the number of tumor cell antigens releasedfrom the tumor cell. Design of an siRNA can be readily determinedaccording to the selected target of the siRNA; methods of siRNA designand down-regulation of genes are known in the art, as exemplified inU.S. Pat. Pub. No. 2003-0198627.

In another embodiment, the therapeutic gene product can be a viralattenuation factor. Antiviral proteins or peptides can be expressed bythe viruses provided herein. Expression of antiviral proteins orpeptides can control viral pathogenicity. Exemplary viral attenuationfactors include, but are not limited to, virus-specific antibodies,mucins, thrombospondin, and soluble proteins such as cytokines,including, but not limited to TNFα, interferons (for example IFNα, IFNβ,or IFNγ) and interleukins (for example IL-1, IL-12 or IL-18).

In another embodiment, the therapeutic gene product can be a proteinligand, such as antitumor oligopeptide. Antitumor oligopeptides areshort protein peptides with high affinity and specificity to tumors.Such oligopeptides could be enriched and identified usingtumor-associated phage libraries (Akita et al. (2006) Cancer Sci.97(10):1075-1081). These oligopeptides have been shown to enhancechemotherapy (U.S. Pat. No. 4,912,199). The oligopeptides can beexpressed by the viruses provided herein. Expression of theoligopeptides can elicit anticancer activities on their own or incombination with other chemotherapeutic agents. An exemplary group ofantitumor oligopeptides is antimitotic peptides, including, but notlimited to, tubulysin (Khalil et al. (2006) Chembiochem. 7(4):678-683),phomopsin, hemiasterlin, taltobulin (HTI-286, 3), and cryptophycin.Tubulysin is from myxobacteria and can induce depletion of cellmicrotubules and trigger the apoptotic process. The antimitotic peptidescan be expressed by the viruses provide herein and elicit anticanceractivities on their own or in combination with other therapeuticmodalities.

In another embodiment, the therapeutic gene product can be a proteinthat sequesters molecules or nutrients needed for tumor growth. Forexample, the virus can express one or more proteins that bind iron,transport iron, or store iron, or a combination thereof. Increased ironuptake and/or storage by expression of such proteins not only, increasescontrast for visualization and detection of a tumor or tissue in whichthe virus accumulates, but also depletes iron from the tumorenvironment. Iron depletion from the tumor environment removes a vitalnutrient from the tumors, thereby deregulating iron hemostasis in tumorcells and delaying tumor progression and/or killing the tumor.

Additionally, iron, or other labeled metals, can be administered to atumor-bearing subject, either alone, or in a conjugated form. An ironconjugate can include, for example, iron conjugated to an imaging moietyor a therapeutic agent. In some cases, the imaging moiety andtherapeutic agent are the same, e.g., a radionuclide. Bacterial cellinternalization of iron in the tumor, wound, area of inflammation orinfection allows the internalization of iron alone, a supplementalimaging moiety, or a therapeutic agent (which can deliver cytotoxicityspecifically to tumor cells or deliver the therapeutic agent fortreatment of the wound, area of inflammation or infection). Thesemethods can be combined with any of the other methods provided herein.

c. Superantigen

The viruses provided herein can be modified to express one or moresuperantigens. Superantigens are antigens that can activate a largeimmune response, often brought about by a large response of T cells. Avariety of superantigens are known in the art including, but not limitedto, diphtheria toxin, staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2,SED, SEE and SEH), Toxic Shock Syndrome Toxin 1, Exfoliating Toxins(EXft), Streptococcal Pyrogenic Exotoxin A, B and C (SPE A, B and C),Mouse Mammary Tumor Virus proteins (MMTV), Streptococcal M proteins,Clostridial Perfringens Enterotoxin (CPET), Listeria monocytogenesantigen p60, and mycoplasma arthritis superantigens.

Since many superantigens also are toxins, if expression of a virus ofreduced toxicity is desired, the superantigen can be modified to retainat least some of its superantigenicity while reducing its toxicity,resulting in a compound such as a toxoid. A variety of recombinantsuperantigens and toxoids of superantigens are known in the art, and canreadily be expressed in the viruses provided herein. Exemplary toxoidsinclude toxoids of diphtheria toxin, as exemplified in U.S. Pat. No.6,455,673 and toxoids of Staphylococcal enterotoxins, as exemplified inU.S. Pat. Pub. No. 20030009015.

d. Gene Product to be Harvested

Exemplary genes expressible by a virus provided herein for the purposeof harvesting include human genes. An exemplary list of genes includesthe list of human genes and genetic disorders authored and edited by Dr.Victor A. McKusick and his colleagues at Johns Hopkins University andelsewhere, and developed for the World Wide Web by NCBI, the NationalCenter for Biotechnology Information. Online Mendelian Inheritance inMan, OMIM™, Center for Medical Genetics, Johns Hopkins University(Baltimore, Md.) and National Center for Biotechnology Information,National Library of Medicine (Bethesda, Md.), and those available inpublic databases, such as PubMed and GenBank (see, for example, genesprovided in the website ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM).

e. Control of Heterologous Gene Expression

In one embodiment, expression the therapeutic compound can be controlledby a regulatory sequence. Suitable regulatory sequences which, forexample, are functional in a mammalian host cell are well known in theart. In one example, the regulatory sequence contains a poxviruspromoter. In another embodiment, the regulatory sequence can contain anatural or synthetic vaccinia virus promoter. Exemplary vaccinia early,intermediate and late stage promoters include, for example, vacciniaP7.5 k early/late promoter, vaccinia P_(EL) early/late promoter,vaccinia P₁₃ early promoter, vaccinia P_(11k) late promoter and vacciniapromoters listed elsewhere herein. Exemplary synthetic promotersinclude, for example, P_(SE) synthetic early promoter, P_(SEL) syntheticearly/late promoter, P_(SL) synthetic late promoter, vaccinia syntheticpromoters listed elsewhere herein (Patel et al., Proc. Natl. Acad. Sci.USA 85: 9431-9435 (1988); Davison and Moss, J Mol Biol 210: 749-769(1989); Davison et al., Nucleic Acids Res. 18: 4285-4286 (1990);Chakrabarti et al., BioTechniques 23: 1094-1097 (1997)). Combinations ofdifferent promoters can be used to express different gene products inthe same virus or two different viruses. In one embodiment, differenttherapeutic or detectable gene products are expressed from differentpromoters, such as two different vaccinia synthetic promoters.

E. METHODS FOR MAKING A MODIFIED VIRUS

The viruses provided herein can be formed by standard methodologies wellknown in the art for modifying viruses. Briefly, the methods includeintroducing into viruses one or more genetic modifications, followed byscreening the viruses for properties reflective of the modification orfor other desired properties.

1. Genetic Modifications

Standard techniques in molecular biology can be used to generate themodified viruses provided herein. Such techniques include variousnucleic acid manipulation techniques, nucleic acid transfer protocols,nucleic acid amplification protocols, and other molecular biologytechniques known in the art. For example, point mutations can beintroduced into a gene of interest through the use of oligonucleotidemediated site-directed mutagenesis. Alternatively, homologousrecombination can be used to introduce a mutation or exogenous sequenceinto a target sequence of interest. In an alternative mutagenesisprotocol, point mutations in a particular gene can also be selected forusing a positive selection pressure. See, e.g., Current Techniques inMolecular Biology, (Ed. Ausubel, et al.). Nucleic acid amplificationprotocols include but are not limited to the polymerase chain reaction(PCR). Use of nucleic acid tools such as plasmids, vectors, promotersand other regulating sequences, are well known in the art for a largevariety of viruses and cellular organisms. Nucleic acid transferprotocols include calcium chloride transformation/transfection,electroporation, liposome mediated nucleic acid transfer,N-[1-(2,3-Dioloyloxy)propyl]-N,N,N-trimethylammonium methylsulfatemeditated transformation, and others. Further a large variety of nucleicacid tools are available from many different sources including ATCC, andvarious commercial sources. One skilled in the art will be readily ableto select the appropriate tools and methods for genetic modifications ofany particular virus according to the knowledge in the art and designchoice.

Any of a variety of modifications can be readily accomplished usingstandard molecular biological methods known in the art. Themodifications will typically be one or more truncations, deletions,mutations or insertions of the viral genome. In one embodiment, themodification can be specifically directed to a particular sequence. Themodifications can be directed to any of a variety of regions of theviral genome, including, but not limited to, a regulatory sequence, to agene-encoding sequence, or to a sequence without a known role. Any of avariety of regions of viral genomes that are available for modificationare readily known in the art for many viruses, including the virusesspecifically listed herein. As a non-limiting example, the loci of avariety of vaccinia genes provided herein and elsewhere exemplify thenumber of different regions that can be targeted for modification in theviruses provided herein. In another embodiment, the modification can befully or partially random, whereupon selection of any particularmodified virus can be determined according to the desired properties ofthe modified the virus. These methods include, for example, in vitrorecombination techniques, synthetic methods and in vivo recombinationmethods as described, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor LaboratoryPress, cold Spring Harbor N.Y. (1989), and in the Examples disclosedherein.

In some embodiments, the virus can be modified to express an exogenousgene. Exemplary exogenous gene products include proteins and RNAmolecules. The modified viruses can express a detectable gene product, atherapeutic gene product, a gene product for manufacturing orharvesting, or an antigenic gene product for antibody harvesting. Thecharacteristics of such gene products are described herein andelsewhere. In some embodiments of modifying an organism to express anexogenous gene, the modification can also contain one or more regulatorysequences to regulate expression of the exogenous gene. As is known inthe art, regulatory sequences can permit constitutive expression of theexogenous gene or can permit inducible expression of the exogenous gene.Further, the regulatory sequence can permit control of the level ofexpression of the exogenous gene. In some examples, inducible expressioncan be under the control of cellular or other factors present in a tumorcell or present in a virus-infected tumor cell. In other examples,inducible expression can be under the control of an administrablesubstance, including IPTG, RU486 or other known induction compounds. Anyof a variety of regulatory sequences are available to one skilled in theart according to known factors and design preferences. In someembodiments, such as gene product manufacture and harvesting, theregulatory sequence can result in constitutive, high levels of geneexpression. In some embodiments, such as anti-(gene product) antibodyharvesting, the regulatory sequence can result in constitutive, lowerlevels of gene expression. In tumor therapy embodiments, a therapeuticprotein can be under the control of an internally inducible promoter oran externally inducible promoter.

In other embodiments, organ or tissue-specific expression can becontrolled by regulatory sequences. In order to achieve expression onlyin the target organ, for example, a tumor to be treated, the foreignnucleotide sequence can be linked to a tissue specific promoter and usedfor gene therapy. Such promoters are well known to those skilled in theart (see e.g., Zimmermann et al., Neuron 12: 11-24 (1994); Vidal et al.,EMBO J. 9: 833-840 (1990); Mayford et al., Cell 81: 891-904 (1995); andPinkert et al., Genes & Dev. 1: 268-76 (1987)).

In some embodiments, the viruses can be modified to express two or moreproteins, where any combination of the two or more proteins can be oneor more detectable gene products, therapeutic gene products, geneproducts for manufacturing or harvesting or antigenic gene products forantibody harvesting. In one embodiment, a virus can be modified toexpress a detectable protein and a therapeutic protein. In anotherembodiment, a virus can be modified to express two or more gene productsfor detection or two or more therapeutic gene products. For example, oneor more proteins involved in biosynthesis of a luciferase substrate canbe expressed along with luciferase. When two or more exogenous genes areintroduced, the genes can be regulated under the same or differentregulatory sequences, and the genes can be inserted in the same ordifferent regions of the viral genome, in a single or a plurality ofgenetic manipulation steps. In some embodiments, one gene, such as agene encoding a detectable gene product, can be under the control of aconstitutive promoter, while a second gene, such as a gene encoding atherapeutic gene product, can be under the control of an induciblepromoter. Methods for inserting two or more genes in to a virus areknown in the art and can be readily performed for a wide variety ofviruses using a wide variety of exogenous genes, regulatory sequences,and/or other nucleic acid sequences.

Methods of producing recombinant viruses are known in the art. Providedherein for exemplary purposes are methods of producing a recombinantvaccinia virus. A recombinant vaccinia virus with an insertion in theF14.5L gene (NotI site of LIVP) can be prepared by the following steps:(a) generating (i) a vaccinia shuttle plasmid containing the modifiedF14.5L gene inserted at restriction site X and (ii) a dephosphorylatedwt VV (VGL) DNA digested at restriction site X; (b) transfecting hostcells infected with PUV-inactivated helper VV (VGL) with a mixture ofthe constructs of (i) and (ii) of step a; and (c) isolating therecombinant vaccinia viruses from the transfectants. One skilled in theart knows how to perform such methods, for example by following theinstructions given in co-pending U.S. application Ser. Nos. 10/872,156and 11/238,025; see also Timiryasova et al. (Biotechniques 31: 534-540(2001)). In one embodiment, restriction site X is a unique restrictionsite. A variety of suitable host cells also are known to the personskilled in the art and include many mammalian, avian and insect cellsand tissues which are susceptible for vaccinia virus infection,including chicken embryo, rabbit, hamster and monkey kidney cells, forexample, HeLa cells, RK₁₃, CV-1, Vero, BSC40 and BSC-1 monkey kidneycells.

2. Screening of Modified Viruses

Modified viruses can be screened for any desired characteristics,including the characteristics described herein such as attenuatedpathogenicity, reduced toxicity, preferential accumulation in tumor,increased ability to activate an immune response against tumor cells,increased immunogenicity, increased or decreased replication competence,and are able to express exogenous proteins, and combinations thereof.For example, the modified viruses can be screened for the ability toactivate an immune response against tumor cells without aggressivelykilling the tumor cells. In another example, the viruses can be screenedfor expression of one or more detectable genes, including genes that canbe used for imaging, or for expression of one or more genes formanufacture or harvest of the gene products and/or for harvest ofantibodies against the gene products.

Any of a variety of known methods for screening for such characteristicscan be performed, as demonstrated in the Examples provided herein. Oneexemplary method for screening for desired characteristics includes, butis not limited to, monitoring growth, replication and/or gene expression(including expression of an exogenous gene) in cell culture or other invitro medium. The cell culture can be from any organism, and from anytissue source, and can include tumorous tissues. Other exemplary methodsfor screening for desired characteristics include, but are not limitedto, administering a virus to animal, including non-human animals such asa mouse, monkey or ape, and optionally also including humans, andmonitoring the virus, the tumor, and or the animal; monitoring can beperformed by in vivo imaging of the virus and/or the tumor (e.g., lowlight imaging of viral gene expression or ultrasonic tumor imaging),external monitoring of the tumor (e.g., external measurement of tumorsize), monitoring the animal (e.g., monitoring animal weight, bloodpanel, antibody titer, spleen size, or liver size). Other exemplarymethods for screening for desired characteristics include, but are notlimited to, harvesting a non-human animal for the effects and locationof the virus and expression by the virus, including methods such asharvesting a variety of organs including a tumor to determine presenceof the virus and/or gene expression by the virus in the organs or tumor,harvesting of organs associated with an immune response or viralclearance such as the spleen or liver, harvesting the tumor to determinetumor size and viability of tumor cells, harvesting antibodies orantibody producing cells. Such screening and monitoring methods can beused in any of a variety of combinations, as is known in art. In oneembodiment, a virus can be screened by administering the virus to ananimal such as a non-human animal or a human, followed by monitoring byin vivo imaging. In another embodiment, a virus can be screened byadministering the virus to an animal such as a non-human animal,monitoring by in vivo imaging, and then harvesting the animal. Thus,provided herein are methods for screening a virus for desiredcharacteristics by administering the virus to an animal such as ananimal with a tumor, and monitoring the animal, tumor (if present),and/or virus in the animal for one or more characteristics. Alsoprovided herein are methods for screening a virus for desiredcharacteristics by administering the virus to a non-human animal such asa non-human animal with a tumor, harvesting the animal, and assaying theanimal's organs, antibody titer, and/or tumor (if present) for one ormore characteristics.

Provided herein are methods for screening a virus for attenuatedpathogenicity or reduced toxicity, where the pathogenicity or toxicitycan be determined by a variety of techniques, including, but not limitedto, assessing the health state of the subject, measuring the body weightof a subject, blood or urine analysis of a subject, and monitoringtissue distribution of the virus within the subject; such techniques canbe performed on a living subject in vivo, or can be performed postmortem. Methods also can include the ability of the viruses to lysecells or cause cell death, which can be determined in vivo or in vitro.

When a subject drops below a threshold body weight, the virus can beconsidered pathogenic to the subject. Exemplary thresholds can be a dropof about 5% or more, a drop of about 10% or more, or a drop of about 15%or more in body weight relative to a reference. A body weight referencecan be selected from any of a variety of references used in the art; forexample, a body weight reference can be the weight of the subject priorto administration of the virus, the body weight reference can be acontrol subject having the same condition as the test subject (e.g.,normal or tumor-injected), where the change in weight of the control iscompared to the change in weight of the test subject for the time periodafter administration of the virus.

Blood or urine analysis of the subject can indicate level of immuneresponse, level of toxins in the subject, or other levels of stress tocells, tissues or organs of the subject such as kidneys, pancreas, liverand spleen. Levels increased above established threshold levels canindicate pathogenicity of the virus to the subject. Threshold levels ofcomponents of blood or urine for indicating viral pathogenicity are wellknown in the art, and any such thresholds can be selected hereinaccording to the desired tolerance of pathogenicity or toxicity of thevirus.

Tissue distribution of a virus in a subject can indicate pathogenicityor toxicity of the virus. In one embodiment, tissue distribution of avirus that is not pathogenic or toxic can be mostly in tumor relative toother tissues or organs. Microorganisms located mostly in tumor canaccumulate, for example, at least about 2-fold greater, at least about5-fold greater, at least about 10-fold greater, at least about 100-foldgreater, at least about 1.000-fold greater, at least about 10.000-foldgreater, at least about 100.000-fold greater, or at least about1,000,000-fold greater, than the viruses accumulate in any otherparticular organ or tissue.

Provided herein are methods for screening a virus for tissuedistribution or accumulation, where the tissue distribution can bedetermined by a variety of techniques, including, but not limited to,harvesting a non-human subject, in vivo imaging a detectable geneproduct in subject. Harvesting can be accomplished by euthanizing thenon-human subject, and determining the accumulation of viruses in tumorand, optionally, the accumulation in one or more additional tissues ororgans. The accumulation can be determined by any of a variety ofmethods, including, but not limited to, detecting gene products such asdetectable gene products (e.g., GFP or beta galactosidase), histologicalor microscopic evaluation of tissue, organ or tumor samples, ormeasuring the number of plaque or colony forming units present in atissue, organ or tumor sample. In one embodiment, the desired amount oftissue distribution of a virus can be mostly in tumor relative to othertissues or organs. Microorganisms located mostly in tumor canaccumulate, for example, at least about 2-fold greater, at least about5-fold greater, at least about 10-fold greater, at least about 100-foldgreater, at least about 1.000-fold greater, at least about 10.000-foldgreater, at least about 100.000-fold greater, or at least about1,000,000-fold greater, than the viruses accumulate in any otherparticular organ or tissue.

Also provided herein are methods of screening for viruses that canelicit an immune response, where the immune response can be against thetumor cells or against the viruses. A variety of methods for measuringthe ability to elicit an immune response are known in the art, andinclude measuring an overall increase in immune activity in a subject,measuring an increase in anti-virus or anti-tumor antibodies in asubject, testing the ability of a virus-treated (typically a non-human)subject to prevent later infection/tumor formation or to rapidlyeliminate viruses or tumor cells. Methods also can include the abilityof the viruses to lyse cells or cause cell death, which can bedetermined in vivo or in vitro.

Also provided herein are methods for determining increased or decreasedreplication competence, by monitoring the speed of replication of theviruses. Such measurements can be performed in vivo or in vitro. Forexample, the speed of replication in a cell culture can be used todetermine replication competence of a virus. In another example, thespeed of replication in a tissue, organ or tumor in a subject can beused to measure replication competence. In some embodiments, decreasedreplication competence in non-tumor tissues and organs can be thecharacteristic to be selected in a screen. In other embodiments,increased replication competence in tumors can be the characteristic tobe selected in a screen.

Also provided herein are methods for determining the ability of a virusto express genes, such as exogenous gene. Such methods can be performedin vivo or in vitro. For example, the viruses can be screened onselective plates for the ability to express a gene that permits survivalof the virus or permits the virus to provide a detectable signal, suchas turning X-gal blue. Such methods also can be performed in vivo, whereexpression can be determined, for example, by harvesting tissues, organsor tumors a non-human subject or by in vivo imaging of a subject.

Also provided herein are methods for determining the ability of a virusto express genes toward which the subject can develop antibodies,including exogenous genes toward which the subject can developantibodies. Such methods can be performed in vivo using any of a varietyof non-human subjects. For example, gene expression can be determined,for example, by bleeding a non-human subject to which a virus has beenadministered, and assaying the blood (or serum) for the presence ofantibodies against the virus-expressed gene, or by any other methodgenerally used for polyclonal antibody harvesting, such as productionbleeds and terminal bleeds.

Also provided herein are methods for screening a virus that has two ormore characteristics provided herein, including screening for attenuatedpathogenicity, reduced toxicity, preferential accumulation in tumor,increased ability to activate an immune response against tumor cells,increased immunogenicity, increased or decreased replication competence,ability to express exogenous proteins, and ability to elicit antibodyproduction against a virally expressed gene product. A single monitoringtechnique, such as in vivo imaging, can be used to verify two or morecharacteristics, or a variety of different monitoring techniques can beused, as can be determined by one skilled in the art according to theselected characteristics and according to the monitoring techniquesused.

Mouse models of different types of human and non-human animal cancerscan be employed to assess the properties of the modified viruses. Tumorscan be established by implantation of different tumor cell types.Exemplary human tumor xenograft models in mice include, but are notlimited to, human lung carcinoma (A549 cells, ATCC No. CCL-185); humanbreast tumor (GI-101A cells, Rathinavelu et al., Cancer Biochem.Biophys., 17:133-146 (1999)); human ovarian carcinoma (OVCAR-3 cells,ATCC No. HTB-161); human pancreatic carcinoma (PANC-1cells, ATCC No.CRL-1469 and MIA PaCa-2 cells, ATCC No. CRL-1420); DU145 cells (humanprostate cancer cells, ATCC No. HTB-81); human prostate cancer (PC-3cells, ATCC# CRL-1435); colon carcinoma (HT-29 cells); human melanoma(888-MEL cells, 1858-MEL cells or 1936-MEL cells; see e.g. Wang et al.,(2006) J. Invest. Dermatol. 126:1372-1377); and human fibrosarcoma(HT-1080 cells, ATCC No. CCL-121,). Exemplary rat tumor xenograft modelsin mice include, but are not limited to, glioma tumor (C6 cells; ATCCNo. CCL-107). Exemplary mouse tumor homograft models include, but arenot limited to, mouse melanoma (B16-F10 cells; ATCC No. CRL-6475).Exemplary cat tumor xenograft models in mice include, but are notlimited to, feline fibrosarcoma (FC77.T cells; ATCC No. CRL-6105).Exemplary dog tumor xenograft models in mice include, but are notlimited to, canine osteosarcoma (D17 cells; ATCC No. CCL-183).

F. VIRUSES FOR USE IN THE METHODS

The viruses provided herein and viruses for the use in the methodprovided typically have one or more of the characteristics providedherein. For example, viruses provided herein can have attenuatedpathogenicity, reduced toxicity, preferential accumulation inimmunoprivileged cells and tissues, such as tumor, ability to activatean immune response against tumor cells, immunogenic, replicationcompetent and are able to express exogenous proteins, and combinationsthereof. The viruses can be RNA or DNA viruses. The viruses can becytoplasmic viruses, such as poxviruses, or can be nuclear viruses suchas adenoviruses. The viruses provided herein can have as part of theirlife cycle lysis of the host cell's plasma membrane. Alternatively, theviruses provided herein can have as part of their life cycle exit of thehost cell by non-lytic pathways such as budding or exocytosis. Theviruses provided herein can cause a host organism to develop an immuneresponse to virus-infected tumor cells as a result of lysis or apoptosisinduced as part of the viral life cycle. The viruses provided hereinalso can be genetically engineered to cause a host organism to developan immune response to virus-infected tumor cells as a result of lysis orapoptosis, regardless of whether or not lysis or apoptosis is induced aspart of the viral life cycle. In some embodiments, the viruses providedherein can cause the host organism to mount an immune response againsttumor cells without lysing or causing cell death of the tumor cells.

One skilled in the art can select from any of a variety of viruses,according to a variety of factors, including, but not limited to, theintended use of the virus, such as a diagnostic and/or therapeutic use(e.g., tumor therapy or diagnosis, vaccination, antibody production, orheterologous protein production), the host organism, and the type oftumor.

The methods provided herein for increasing or decreasing the attenuationof a virus are typically applied to a virus that is used for adiagnostic or therapeutic use in a subject (i.e., a therapeutic virus).A therapeutic virus for the methods provided herein can exhibit one ormore desired characteristics for use as a therapeutic agent, such as,for example attenuated pathogenicity, reduced toxicity, preferentialaccumulation in immunoprivileged cells and tissues, such as tumor,ability to activate an immune response against tumor cells, immunogenic,replication competent, and are able to express exogenous proteins, andcombinations thereof.

1. Cytoplasmic Viruses

The viruses provided herein can be cytoplasmic viruses, where the lifecycle of the virus does not require entry of viral nucleic acidmolecules in to the nucleus of the host cell. A variety of cytoplasmicviruses are known, including, but not limited to, pox viruses, Africanswine flu family viruses, and various RNA viruses such aspicornaviruses, caliciviruses, togaviruses, coronaviruses andrhabdoviruses. In some embodiments, viral nucleic acid molecules do notenter the host cell nucleus throughout the viral life cycle. In otherembodiments, the viral life cycle can be performed without use of hostcell nuclear proteins. In other embodiments, the virulence orpathogenicity of the virus can be modulated by modulating the activityof one or more viral proteins involved in viral replication.

a. Poxviruses

In one embodiment, the virus provided herein is selected from thepoxvirus family. Mechanisms for the control of transcription areconserved across the members of the poxvirus family (Broyles et al. J.Gen. Virol (2003) 84: 2293-2303). Poxviruses include Chordopoxyiridaesuch as orthopoxvirus, parapoxvirus, avipoxvirus, capripoxvirus,leporipoxvirus, suipoxvirus, molluscipoxvirus and yatapoxvirus, as wellas Entomopoxyirinae such as entomopoxvirus A, entomopoxvirus B, andentomopoxvirus A. Chordopoxyiridae are vertebrate poxviruses and havesimilar antigenicities, morphologies and host ranges; thus, any of avariety of such poxviruses can be used herein. One skilled in the artcan select a particular genera or individual chordopoxyiridae accordingto the known properties of the genera or individual virus, and accordingto the selected characteristics of the virus (e.g., pathogenicity,ability to elicit and immune response, preferential tumor localization),the intended use of the virus, the tumor type and the host organism.Exemplary chordopoxyiridae genera are orthopoxvirus and avipoxvirus.

Avipoxviruses are known to infect a variety of different birds and havebeen administered to humans. Exemplary avipoxviruses include canarypox,fowlpox, juncopox, mynahpox, pigeonpox, psittacinepox, quailpox,peacockpox, penguinpox, sparrowpox, starlingpox and turkeypox viruses.

Orthopoxviruses are known to infect a variety of different mammalsincluding rodents, domesticated animals, primates and humans. Severalorthopoxviruses have a broad host range, while others have narrower hostrange. Exemplary orthopoxviruses include buffalopox, camelpox, cowpox,ectromelia, monkeypox, raccoon pox, skunk pox, tatera pox, uasin gishu,vaccinia, variola, and volepox viruses. In some embodiments, theorthopoxvirus selected can be an orthopoxvirus known to infect humans,such as cowpox, monkeypox, vaccinia, or variola virus. Optionally, theorthopoxvirus known to infect humans can be selected from the group oforthopoxviruses with a broad host range, such as cowpox, monkeypox, orvaccinia virus.

i. Vaccinia Virus

One exemplary orthopoxvirus presented in the methods provided herein isvaccinia virus. A variety of vaccinia virus strains are available,including Western Reserve (WR), Copenhagen, Tashkent, Tian Tan, Lister,Wyeth, 1HD-J, and IHD-W, Brighton, Ankara, MVA, Dairen I, LIPV, LC16M8,LC16MO, LIVP, WR 65-16, Connaught, New York City Board of Health.Exemplary vaccinia viruses are Lister or LIVP vaccinia viruses. In oneembodiment, the Lister strain can be an attenuated Lister strain, suchas the LIVP (Lister virus from the Institute of Viral Preparations,Moscow, Russia) strain, which was produced by further attenuation of theLister strain. The LIVP strain was used for vaccination throughout theworld, particularly in India and Russia, and is widely available. Inanother embodiment, the viruses and methods provided herein can be basedon modifications to the Lister strain of vaccinia virus. Lister (alsoreferred to as Elstree) vaccinia virus is available from any of avariety of sources. For example, the Elstree vaccinia virus is availableat the ATCC under Accession Number VR-1549. The Lister vaccinia strainhas high transduction efficiency in tumor cells with high levels of geneexpression.

Any known vaccinia virus, or modifications thereof that correspond tothose provided herein or known to those of skill in the art to reducetoxicity of a vaccinia virus. Generally, however, the mutation will be amultiple mutant and the virus will be further selected to reducetoxicity.

Vaccinia virus possesses a variety of features for use in cancer genetherapy and vaccination. It has a broad host and cell type range.Vaccinia is a cytoplasmic virus, thus, it does not insert its genomeinto the host genome during its life cycle.

The linear dsDNA viral genome of vaccinia virus is approximately 200 kbin size, encoding a total of approximately 200 potential genes. Thevaccinia virus genome has a large carrying capacity for foreign genes,where up to 25 kb of exogenous DNA fragments (approximately 12% of thevaccinia genome size) can be inserted. The genomes of several of thevaccinia strains have been completely sequenced, and many essential andnonessential genes identified. Due to high sequence homology amongdifferent strains, genomic information from one vaccinia strain can beused for designing and generating modified viruses in other strains.Finally, the techniques for production of modified vaccinia strains bygenetic engineering are well established (Moss, Curr. Opin. Genet. Dev.3: 86-90 (1993); Broder and Earl, Mol. Biotechnol. 13: 223-245 (1999);Timiryasova et al., Biotechniques 31: 534-540 (2001)).

Historically, vaccinia virus was used to immunize against smallpoxinfection. More recently, modified vaccinia viruses are being developedas vaccines to combat a variety of diseases. Attenuated vaccinia viruscan trigger a cell-mediated immune response. Strategies such asprime/boost vaccination, vaccination with nonreplicating vaccinia virusor a combination of these strategies, have shown promising results forthe development of safe and effective vaccination protocols. Mutantvaccinia viruses from previous studies exhibit a variety ofshortcomings, including a lack of efficient delivery of the viralvehicle to the desired tissue only (e.g., specific accumulation in atumors), a lack of safety because of possible serious complications(e.g., in young children, eczema vaccinatum and encephalitis, and inadults disseminated or progressive vaccinia can result if the individualis severely immunodeficient).

During the vaccinia life cycle, transcription of vaccinia genes occursin three stages: early, intermediate, and late, which correspond to thestages of viral replication and virion assembly. Progression througheach stage occurs by coordinated involvement of viral and host proteins.Early stage gene expression depends on viral transcription factorslocated within the virion core, whereas late gene expression requiresthe cooperation of host proteins and viral factors, including newlyexpressed viral transcription factors. Exemplary of poxvirus early genesinclude those that encode proteins involved in evasion of host defenses,DNA replication, nucleotide biosynthesis, and intermediate genetranscription. Exemplary intermediate and late genes include those thatencode factors needed for late gene expression and proteins involved invirion morphogenesis and assembly. In addition, several vaccinia genesare continuously transcribed throughout infection.

Increases in promoter competition can be generated by competition forendogenous vaccinia transcription, but can also involve host factors aswell. Studies have shown the involvement of host cellular proteins inthe intermediate and late stages of vaccinia viral transcription. Forexample, reconstitution experiments for studying vaccinia intermediatetranscription in vitro indicated the requirement for one or morecellular factors located in the nuclear fraction, and additionally, inthe cytoplasm of infected cells (Rosales et el. (1994) Proc. Natl. Acad.Sci. USA 91:3794-3798). Ribonucleoproteins, such as A2/B1 and RBM3 werealso found to activate transcription vaccinia late promoters (Wright etal. (2001) J. Biol. Chem. 276:40680-40686, Dellis et al. (2004) Virology329(2):328-336). Host cell nuclear proteins, such as YinYang1 (YY1),SP1, and TATA binding protein (TBP) were subsequently found to berecruited from the nucleus to sites of vaccinia viral transcription inthe cytoplasm (Slezak et al. (2004) Virus Res. 102(2):177-184, Oh andBroyles, (2005) J. Virol. 79 (20) 12852-12860). TATA boxes, which bindto TBP, are located in many intermediate and late viral promoters,suggesting a role for this host factor in facilitating the recruitmentof transcription factors to the vaccinia viral promoters. The formationof such TBP-associated complexes can furthermore aid in transcriptionalswitching from early to late viral genes (Knutson et al. (2006) 80(14)6784-6793). Binding sites for YY1 are located downstream of theconserved TAAAT late promoter motif in vaccinia late promoters. YY1,which is a zinc finger transcription factor of the krüppel family, isinvolved in the regulation of cellular genes by acting as an initiatorelement factor that promotes transcription (Shi et al. (1997) Biochim.Biophys. Acta 1332: F49-F66). Data on vaccinia virus suggests that YY1can play a similar role in vaccinia intermediate and late transcription(Broyles et al. (1999) J. Biol. Chem. 274(50):35662-35667). Furthermore,YY1 has been shown to be required for transcription in other viruses,such as, for example, herpesviruses, papillomaviruses polyomaviruses,adenoviruses, parvoviruses, and retroviruses (Chen et al. (1991) J.Virol. 66:4303-4314, Bell et al., (1998) Virology 252:149-161, Bauknechtet al. (1992) EMBO J. 11:4607-4617, Pajunnk et al. (1997) J. Gen. Virol.78:3287-3295, Martelli et al. (1996) J. Virol. 70:1433-1438, Zock et al.(1993) J. Virol. 67:682-693, Momoeda et al. J. Virol. 68:7159-7168, andKnossi et al. (1999) J. Virol. 73:1254-1261).

In addition to the examples provided herein, the effects of geneexpression cassettes on the vaccinia virus growth have been reported inscreens for vaccinia essential genes. In one such study, insertion of anexpression cassette encoding the E. coli guanine phosphoribosyltransferase gene under the control of the vaccinia 7.5K early promoterin the vaccinia F11L gene had a negative effect on viral growth, whereasa point mutation in the F 11L gene did not affect viral growth (Kato etal., (2004) J. Virol. Methods 115(1):31-40). Thus the insertedexpression cassettes encoding non-therapeutic marker genes cancontribute to the overall attenuation of the virus.

Provided herein are vaccinia viruses with insertions, mutations ordeletions, as described more generally elsewhere herein. The vacciniaviruses are modified or selected to have low toxicity and to accumulatein the target tissue. Exemplary of such viruses are those from the LIVPstrain.

Exemplary insertions, mutations or deletions are those that result in anattenuated vaccinia virus relative to the wild type strain. For example,vaccinia virus insertions, mutations or deletions can decreasepathogenicity of the vaccinia virus, for example, by reducing thetoxicity, reducing the infectivity, reducing the ability to replicate,or reducing the number of non-tumor organs or tissues to which thevaccinia virus can accumulate. Other exemplary insertions, mutations ordeletions include, but are not limited to, those that increaseantigenicity of the microorganism, those that permit detection orimaging, those that increase toxicity of the microorganism (optionally,controlled by an inducible promoter). For example, modifications can bemade in genes that are involved in nucleotide metabolism, hostinteractions and virus formation. Any of a variety of insertions,mutations or deletions of the vaccinia virus known in the art can beused herein, including insertions, mutations or deletions of: thethymidine kinase (TK) gene, the hemagglutinin (HA) gene, the F14.5L gene(see e.g., U.S. Patent Pub. No. 2005-0031643), the VGF gene (see e.g.,U.S. Pat. Pub. No. 20030031681); a hemorrhagic region or an A typeinclusion body region (see e.g., U.S. Pat. No. 6,596,279); HindIII F,F13L, or HindIII M (see e.g., U.S. Pat. No. 6,548,068); A33R, A34R, A36Ror B5R genes (see, e.g., Katz et al., J. Virology 77:12266-12275(2003)); SalF7L (see, e.g., Moore et al., EMBO J. 1992 11:1973-1980);NIL (see, e.g., Kotwal et al., Virology 171: 579-587 (1989)); M1 lambda(see, e.g., Child et al., Virology. 174: 625-629 (1990)); HR,HindIII-MK, HindIII-MKF, HindIII-CNM, RR, or BamF (see, e.g., Lee etal., J. Virol. 66: 2617-2630 (1992)); or C21L (see, e.g., Isaacs et al.,Proc. Natl. Acad. Sci. USA. 89: 628-632 (1992)).

Modification of vaccinia viruses at F14.5L gene is described in U.S.Patent Pub. No. 2005-0031643 (referred to as F3 gene therein; see alsoMikryukov et al. (1988) Biotekhnologiya 4: 442-449). For example, theF14.5L gene has been modified at the unique single NotI restriction sitelocated within the F14.5L gene at position 35 or at position 1475 insideof the HindIII-F fragment of vaccinia virus DNA strain LIVP (Mikryukovet al., Biotekhnologiy 4: 442-449 (1988)) by insertion of a foreign DNAsequence into the NotI digested virus DNA. Thus, for use in the methodsprovided herein, vaccinia viruses can contain an insertion, mutation ordeletion of the F14.5L gene or a mutation of a corresponding locus. Invaccinia virus strain Copenhagen (Goebel et al., Virology 179: 247-266(1990)) the NotI restriction site is located between the two openreading frames (ORF) encoding F14L and F15L genes. In vaccinia virusstrain LIVP, the NotI restriction site is located in the ORF encodingthe F14.5L gene with unknown function (Mikryukov et al., Biotekhnologiya4: 442-449 (1988)). Results of the animal experiments suggest thatinterruption of the F14.5L gene with a gene expression cassette iscorrelated with decreased viral virulence, though it is not knownwhether mutation of the F14.5L gene itself contributes to the decreasein virulence.

The F14.5L gene is conserved in a variety of different vaccinia virusstrains, including WR (nucleotides 42238-42387 of GenBank Accession No.AY243312.1, Ankara (nucleotides 37155-37304 of GenBank Accession No.U94848.1), Tian Tan (nucleotides 41808-41954 of GenBank Accession No.AF095689), Acambis 3000 (nucleotides 31365-31514 of GenBank AccessionNo. AY603355.1) and Copenhagen (nucleotides 45368-45517 of GenBankAccession No. M35027.1) strains. The F3 gene also is conserved in thelarger family of poxviruses, particularly among orthopoxviruses such ascowpox (nucleotides 58498-58647 of GenBank Accession No. X94355.2),rabbitpox (nucleotides 46969-47118 of GenBank Accession No. AY484669.1),camelpox (nucleotides 43331-43480 of GenBank Accession No. AY009089.1),ectromelia (nucleotides 51008-51157 of GenBank Accession No.AF012825.2), monkeypox (nucleotides 42515-42660 of GenBank Accession No.AF380138.1), and variola viruses (nucleotides 33100-33249 of GenBankAccession No. X69198.1). Accordingly, also provided are modifications ofthe equivalent of the F14.5L gene in poxviruses, such as orthopoxvirusesincluding a variety of vaccinia virus strains. One skilled in the artcan identify the location of the equivalent F14.5L gene in a variety ofpoxviruses, orthopoxviruses and vaccinia viruses. In another example,the equivalent to the F14.5L gene in LIVP can be determined by itsstructural location in the viral genome: the F3 gene is located on theHindIII-F fragment of vaccinia virus between open reading frames F14Land F15L as defined by Goebel et al. (Virology 179: 247-266 (1990)), andin the opposite orientation of ORFs F14L and F15L; one skilled in theart can readily identify the gene located in the structurally equivalentregion in a large variety of related viruses, such as a large variety ofpox viruses.

Comparative protein sequence analysis revealed some insight into proteinfunction. The closest match with the protein encoded by the F14.5L gene(strain LIVP) is a prolyl 4-hydroxylase alpha subunit precursor (4-PHalpha) from the nematode Caenorhabditis elegans (Veijola et al., J.Biol. Chem. 269: 26746-26753 (1994)). This alpha subunit forms an activealpha-beta dimer with the human protein disulfide isomerase betasubunit. Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of4-hydroxyproline in collagen. The vertebrate enzyme is an alpha 2-beta 2tetramer, the beta subunit of which is identical to the proteindisulfide-isomerase (PDI). However, the importance of this protein forvaccinia viral replication is unknown.

b. Other Cytoplasmic Viruses

Also provided herein are cytoplasmic viruses that are not poxviruses.Cytoplasmic viruses can replicate without introducing viral nucleic acidmolecules into the nucleus of the host cell. A variety of suchcytoplasmic viruses are known in the art, and include African swine flufamily viruses and various RNA viruses such as arenaviruses,picornaviruses, caliciviruses, togaviruses, coronaviruses,paramyxoviruses, flaviviruses, reoviruses, and rhaboviruses. Exemplarytogaviruses include Sindbis viruses. Exemplary arenaviruses includelymphocytic choriomeningitis virus. Exemplary rhaboviruses includevesicular stomatitis viruses. Exemplary paramyxoviruses includeNewcastle Disease viruses and measles viruses. Exemplary picornavirusesinclude polio viruses, bovine enteroviruses and rhinoviruses. Exemplaryflaviviruses include Yellow fever virus; attenuated Yellow fever virusesare known in the art, as exemplified in Barrett et al. (Biologicals 25:17-25 (1997)), and McAllister et al. (J. Virol. 74: 9197-9205 (2000)).

Also provided herein are modifications of the viruses provided above toenhance one or more characteristics relative to the wild type virus.Such characteristics can include, but are not limited to, attenuatedpathogenicity, reduced toxicity, preferential accumulation in tumor,increased ability to activate an immune response against tumor cells,increased immunogenicity, increased or decreased replication competence,and are able to express exogenous proteins, and combinations thereof. Insome embodiments, the modified viruses have an ability to activate animmune response against tumor cells without aggressively killing thetumor cells. In other embodiments, the viruses can be modified toexpress one or more detectable genes, including genes that can be usedfor imaging. In other embodiments, the viruses can be modified toexpress one or more genes for harvesting the gene products and/or forharvesting antibodies against the gene products.

2. Adenovirus, Herpes, Retroviruses

Further provided herein are viruses that include in their life cycleentry of a nucleic acid molecule into the nucleus of the host cell. Avariety of such viruses is known in the art, and includes herpesviruses,papovaviruses, retroviruses, adenoviruses, parvoviruses andorthomyxoviruses. Exemplary herpesviruses include herpes simplex type Iviruses, cytomegaloviruses, and Epstein-Barr viruses. Exemplarypapovaviruses include human papillomavirus and SV40 viruses. Exemplaryretroviruses include lentiviruses. Exemplary orthomyxoviruses includeinfluenza viruses. Exemplary parvoviruses include adeno associatedviruses.

Also provided herein are modifications of the viruses provided above toenhance one or more characteristics relative to the wild type virus.Such characteristics can include, but are not limited to, attenuatedpathogenicity, reduced toxicity, preferential accumulation in tumor,increased ability to activate an immune response against tumor cells,increased immunogenicity, increased or decreased replication competence,and are able to express exogenous proteins, and combinations thereof. Insome embodiments, the modified viruses have an ability to activate animmune response against tumor cells without aggressively killing thetumor cells. In other embodiments, the viruses can be modified toexpress one or more detectable genes, including genes that can be usedfor imaging. In other embodiments, the viruses can be modified toexpress one or more genes for harvesting the gene products and/or forharvesting antibodies against the gene products.

G. EXEMPLARY CHARACTERISTICS OF THE VIRUSES

The viruses provided herein, viruses provided for use in the methods,and viruses that have been modified using the methods provided hereincan accumulate in immunoprivileged cells or immunoprivileged tissues,including tumors and/or metastases, and also including wounded tissuesand cells. While the viruses provided herein can typically be clearedfrom the subject to whom the viruses are administered by activity of thesubject's immune system, viruses can nevertheless accumulate, surviveand proliferate in immunoprivileged cells and tissues such as tumorsbecause such immunoprivileged areas are sequestered from the host'simmune system. Accordingly, the methods provided herein, as applied totumors and/or metastases, and therapeutic methods relating thereto, canreadily be applied to other immunoprivileged cells and tissues,including wounded cells and tissues.

1. Attenuated

The viruses provided herein and viruses provided for use in the methodsare typically attenuated. Attenuated viruses have a decreased capacityto cause disease in a host. The decreased capacity can result from anyof a variety of different modifications to the ability of a virus to bepathogenic. For example, a virus can have reduced toxicity, reducedability to accumulate in non-tumorous organs or tissue, reduced abilityto cause cell lysis or cell death, or reduced ability to replicatecompared to the non-attenuated form thereof. The attenuated virusesprovided herein, however, retain at least some capacity to replicate andto cause immunoprivileged cells and tissues, such as tumor cells to leakor lyse, undergo cell death, or otherwise cause or enhance an immuneresponse to immunoprivileged cells and tissues, such as tumor cells.

a. Reduced Toxicity

Viruses can be toxic to their hosts by manufacturing one or morecompounds that worsen the health condition of the host. Toxicity to thehost can be manifested in any of a variety of manners, including septicshock, neurological effects, or muscular effects. The viruses providedherein can have a reduced toxicity to the host. The reduced toxicity ofa virus of the present methods and compositions can range from atoxicity in which the host experiences no toxic effects, to a toxicityin which the host does not typically die from the toxic effects of themicrobes. In some embodiments, the viruses are of a reduced toxicitysuch that a host typically has no significant long-term effect from thepresence of the viruses in the host, beyond any effect on tumorous,metastatic or necrotic organs or tissues. For example, the reducedtoxicity can be a minor fever or minor infection, which lasts for lessthan about a month, and following the fever or infection, the hostexperiences no adverse effects resultant from the fever or infection. Inanother example, the reduced toxicity can be measured as anunintentional decline in body weight of about 5% or less for the hostafter administration of the microbes. In other examples, the virus hasno toxicity to the host.

b. Accumulate in Tumor, not Substantially in Other Organs

Viruses can accumulate in any of a variety of tissues and organs of thehost. Accumulation can be evenly distributed over the entire hostorganism, or can be concentrated in one or a few organs or tissues. Theviruses provided herein can accumulate in targeted tissues, such asimmunoprivileged cells and tissues, such as tumors and also metastases.In some embodiments, the viruses provided herein exhibit accumulation inimmunoprivileged cells and tissues, such as tumor cells relative tonormal organs or tissues that is equal to or greater than theaccumulation that occurs with wild-type viruses. In other embodiments,the viruses provided herein exhibit accumulation in immunoprivilegedcells and tissues, such as tumor cells that is equal to or greater thanthe accumulation in any other particular organ or tissue. For example,the viruses provided herein can demonstrate an accumulation inimmunoprivileged cells and tissues, such as tumor cells that is at leastabout 2-fold greater, at least about 5-fold greater, at least about10-fold greater, at least about 100-fold greater, at least about1.000-fold greater, at least about 10.000-fold greater, at least about100.000-fold greater, or at least about 1,000,000-fold greater, than theaccumulation in any other particular organ or tissue.

In some embodiments, a virus can accumulate in targeted tissues andcells, such as immunoprivileged cells and tissues, such as tumor cells,without accumulating in one or more selected tissues or organs. Forexample, a virus can accumulate in tumor cells without accumulating inthe brain. In another example, a virus can accumulate in tumor cellswithout accumulating in neural cells. In another example, a virus canaccumulate in tumor cells without accumulating in ovaries. In anotherexample, a virus can accumulate in tumor cells without accumulating inthe blood. In another example, a virus can accumulate in tumor cellswithout accumulating in the heart. In another example, a virus canaccumulate in tumor cells without accumulating in the bladder. Inanother example, a virus can accumulate in tumor cells withoutaccumulating in testes. In another example, a virus can accumulate intumor cells without accumulating in the spleen. In another example, avirus can accumulate in tumor cells without accumulating in the lungs.

One skilled in the art can determine the desired capability for theviruses to selectively accumulate in targeted tissue or cells, such asin an immunoprivileged cells and tissues, such as tumor rather thannon-target organs or tissues, according to a variety of factors known inthe art, including, but not limited to, toxicity of the viruses, dosage,tumor to be treated, immunocompetence of host, and disease state of thehost.

c. Ability to Elicit or Enhance Immune Response to Tumor Cells

Viruses herein can cause or enhance an immune response to antigens inthe targeted tissues or cells, such as immunoprivileged cells andtissues, such as tumor cells. The immune response can be triggered byany of a variety of mechanisms, including the presence or expression ofimmunostimulatory cytokines and the expression or release antigeniccompounds that can cause an immune response.

Cells, in response to an infection such as a viral infection, can sendout signals to stimulate an immune response against the cells. Exemplarysignals sent from such cells include antigens, cytokines and chemokinessuch as interferon-gamma and interleukin-15. The viruses provided hereincan cause targeted cells to send out such signals in response toinfection by the microbes, resulting in a stimulation of the host'simmune system against the targeted cells or tissues, such as tumorcells.

In another embodiment, targeted cells or tissues, such as tumor cells,can contain one or more compounds that can be recognized by the host'simmune system in mounting an immune response against a tumor. Suchantigenic compounds can be compounds on the cell surface or the tumorcell, and can be protein, carbohydrate, lipid, nucleic acid, orcombinations thereof. Viral-mediated release of antigenic compounds canresult in triggering the host's immune system to mount an immuneresponse against the tumor. The amount of antigenic compound released bythe tumor cells is any amount sufficient to trigger an immune responsein a subject; for example, the antigenic compounds released from one ormore tumor cells can trigger a host immune response in the organism thatis known to be accessible to leukocytes.

The time duration of antigen release is an amount of time sufficient forthe host to establish an immune response to one or more tumor antigens.In some embodiments, the duration is an amount of time sufficient forthe host to establish a sustained immune response to one or more tumorantigens. One skilled in the art can determine such a time durationbased on a variety of factors affecting the time duration for a subjectto develop an immune response, including the level of the tumor antigenin the subject, the number of different tumor antigens, the antigenicityof the antigen, the immunocompetence of the host, and the access of theantigenic material to the vasculature of the host. Typically, theduration of antigen release can be at least about a week, at least about10 days, at least about two weeks, or at least about a month.

The viruses provided herein can have any of a variety of properties thatcan cause target cells and tissues, such as tumor cells, to releaseantigenic compounds. Exemplary properties are the ability to lyse cellsand the ability to elicit apoptosis in tumor cells. Viruses that areunable to lyse tumor cells or cause tumor cell death can nevertheless beused in the methods provided herein when such viruses can cause somerelease or display of antigenic compounds from tumor cells. A variety ofmechanisms for antigen release or display without lysis or cell deathare known in the art, and any such mechanism can be used by the virusesprovided herein, including, but not limited to, secretion of antigeniccompounds, enhanced cell membrane permeability, expression ofimmunostimulatory proteins or altered cell surface expression or alteredMHC presentation in tumor cells when the tumor cells can be accessed bythe host's immune system. Regardless of the mechanism by which thehost's immune system is activated, the net result of the presence of theviruses in the tumor is a stimulation of the host's immune system, atleast in part, against the tumor cells. In one example, the viruses cancause an immune response against tumor cells not infected by theviruses.

In one embodiment, the viruses provided herein can cause tumor cells torelease an antigen that is not present on the tumor cell surface. Tumorcells can produce compounds such as proteins that can cause an immuneresponse; however, in circumstances in which the antigenic compound isnot on the tumor cell surface, the tumor can proliferate, and evenmetastasize, without the antigenic compound causing an immune response.Within the scope of the present methods, the viruses provided herein cancause antigenic compounds within the cell to release away from the celland away from the tumor, which can result in triggering an immuneresponse to such an antigen. Even if not all cells of a tumor arereleasing antigens, the immune response can initially be targeted towardthe “leaky” tumor cells, and the bystander effect of the immune responsecan result in further tumor cell death around the “leaky” tumor cells.

d. Balance of Pathogenicity and Release of Tumor Antigens

Typical methods of involving treatment of targeted cells and tissues,such as immunoprivileged cells and tissues, such as tumors, are designedto cause rapid and complete removal thereof. For example, many virusescan cause lysis and/or apoptosis in a variety of cells, including tumorcells. Viruses that can vigorously lyse or cause cell death can behighly pathogenic, and can even kill the host. Furthermore, therapeuticmethods based upon such rapid and complete lysis are typicallytherapeutically ineffective.

In contrast, the viruses provided herein are not aggressive in causingcell death or lysis. They can have a limited or no ability to cause celldeath as long as they accumulate in the target cells or tissues andresult in alteration of cell membranes to cause leakage of antigensagainst which an immune response is mounted. It is desirable that theirapoptotic or lytic effect is sufficiently slow or ineffective to permitsufficient antigenic leakage for a sufficient time for the host to mountan effective immune response against the target tissues. Such immuneresponse alone or in combination with the lytic/apoptotic effect of thevirus results in elimination of the target tissue and also eliminationof future development, such as metastases and reoccurrence, of suchtissues or cells. While the viruses provided herein can have a limitedability to cause cell death, the viruses provided herein cannevertheless stimulate the host's immune system to attack tumor cells.As a result, such viruses also are typically unlikely to havesubstantial toxicity to the host.

In one embodiment, the viruses have a limited, or no ability to causetumor cell death, while still causing or enhancing an immune responseagainst tumor cells. In one example, the rate of viral-mediated tumorcell death is less than the rate of tumor cell growth or replication. Inanother example, the rate of viral-mediated tumor cell death is slowenough for the host to establish a sustained immune response to one ormore tumor antigens. Typically, the time for cell death is sufficient toestablish an anti-tumor immune response and can be at least about aweek, at least about 10 days, at least about two weeks, or at leastabout a month, depending upon the host and the targeted cells ortissues.

In another embodiment, the viruses provided herein can cause cell deathin tumor cells, without causing substantial cell death in non-tumortissues. In such an embodiment, the viruses can aggressively kill tumorcells, as long as no substantial cell death occurs in non-tumor cells,and optionally, so long as the host has sufficient capability to mountan immune response against the tumor cells.

In one embodiment, the ability of the viruses to cause cell death isslower than the host's immune response against the viruses. The abilityfor the host to control infection by the viruses can be determined bythe immune response (e.g., antibody titer) against viral antigens.Typically, after the host has mounted immune response against theviruses, the viruses can have reduced pathogenicity in the host. Thus,when the ability of the viruses to cause cell death is slower than thehost's immune response against the microbes, viral-mediated cell deathcan occur without risk of serious disease or death to the host. In oneexample, the ability of the viruses to cause tumor cell death is slowerthan the host's immune response against the microbes.

2. Immunogenicity

The viruses provided herein also can be immunogenic. An immunogenicvirus can create a host immune response against the virus. In oneembodiment, the viruses can be sufficiently immunogenic to result in alarge anti-viral antibody titer. The viruses provided herein can havethe ability to elicit an immune response. The immune response can beactivated in response to viral antigens or can be activated as a resultof viral-infection induced cytokine or chemokine production. Immuneresponse against the viruses can decrease the likelihood ofpathogenicity toward the host organism.

Immune response against the viruses also can result in target tissue orcell, such as tumor cell, killing. In one embodiment, the immuneresponse against viral infection can result in an immune responseagainst tumor cells, including developing antibodies against tumorantigens. In one example, an immune response mounted against the viruscan result in tumor cell killing by the “bystander effect,” whereuninfected tumor cells nearby infected tumor cells are killed at thesame time as infected cells, or alternatively, where uninfected tumorcells nearby extracellular viruses are killed at the same time as theviruses. As a result of bystander effect tumor cell death, tumor cellantigens can be released from cells, and the host organism's immunesystem can mount an immune response against tumor cell antigens,resulting in an immune response against the tumor itself.

In one embodiment, the virus can be selected or modified to express oneor more antigenic compounds, including superantigenic compounds. Theantigenic compounds such as superantigens can be endogenous geneproducts or can be exogenous gene products. Superantigens, includingtoxoids, are known in the art and described elsewhere herein.

3. Replication Competent

The viruses provided herein can be replication competent. In a varietyof viral systems, the administered virus is rendered replicationincompetent to limit pathogenicity risk to the host. While replicationincompetence can protect the host from the virus, it also limits theability of the virus to infect and kill tumor cells, and typicallyresults in only a short-lived effect. In contrast, the viruses providedherein can be attenuated but replication competent, resulting in lowtoxicity to the host and accumulation mainly or solely in tumors. Thus,the viruses provided herein can be replication competent withoutcreating a pathogenicity risk to the host.

Attenuation of the viruses provided herein can include, but is notlimited to, reducing the replication competence of the virus. Forexample, a virus can be modified to decrease or eliminate an activityrelated to replication, such as a transcriptional activator thatregulates replication in the virus. In an example, a virus, can have theviral thymidine kinase (TK) gene modified, which decreases replicationof the virus.

4. Genetic Variants

The viruses provided herein can be modified from their wild type form.Modifications can include any of a variety of changes, and typicallyinclude changes to the genome or nucleic acid molecules of the viruses.Exemplary nucleic acid molecular modifications include truncations,insertions, deletions and mutations. In an exemplary modification, aviral gene can be modified by truncation, insertion, deletion ormutation. In an exemplary insertion, an exogenous gene can be insertedinto the genome of the virus.

Modifications of the viruses provided herein can result in amodification of viral characteristics, including those provided hereinsuch as pathogenicity, toxicity, ability to preferentially accumulate intumor, ability to lyse cells or cause cell death, ability to elicit animmune response against tumor cells, immunogenicity, and replicationcompetence. Variants can be obtained by general methods such asmutagenesis and passage in cell or tissue culture and selection ofdesired properties, as is known in the art, as exemplified forrespiratory syncytial virus in Murphy et al., Virus Res. 1994, 32:13-26.

Variants also can be obtained by mutagenic methods in which nucleic acidresidues of the virus are added, removed or modified relative to thewild type. Any of a variety of known mutagenic methods can be used,including recombination-based methods, restriction endonuclease-basedmethods, and PCR-based methods. Mutagenic methods can be directedagainst particular nucleotide sequences such as genes, or can be random,where selection methods based on desired characteristics can be used toselect mutated viruses. Any of a variety of viral modifications can bemade, according to the selected virus and the particular knownmodifications of the selected virus.

H. PHARMACEUTICAL COMPOSITIONS, COMBINATIONS AND KITS

Provided herein are pharmaceutical compositions, combinations and kitscontaining a virus provided herein and one or more components.Pharmaceutical compositions can include a virus provided herein and apharmaceutical carrier. Combinations can include two or more viruses, avirus and a detectable compound, a virus and a viral expressionmodulating compound, a virus and a therapeutic compound, or anycombination thereof. Kits can include the pharmaceutical compositionsand/or combinations provided herein, and one or more components, such asinstructions for use, a device for detecting a virus in a subject, adevice for administering a compound to a subject, and a device foradministering a compound to a subject.

1. Pharmaceutical Compositions

Provided herein are pharmaceutical compositions containing a virusprovided herein and a suitable pharmaceutical carrier. Pharmaceuticalcompositions provided herein can be in various forms, e.g., in solid,liquid, powder, aqueous, or lyophilized form. Examples of suitablepharmaceutical carriers are known in the art and include but are notlimited to water, buffers, saline solutions, phosphate buffered salinesolutions, various types of wetting agents, sterile solutions, alcohols,gum arabic, vegetable oils, benzyl alcohols, gelatin, glycerin,carbohydrates such as lactose, sucrose, amylose or starch, magnesiumstearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, pentaerythritol fatty acid esters,hydroxy methylcellulose, powders, among others. Pharmaceuticalcompositions provided herein can contain other additives including, forexample, antioxidants and preservatives, analgesic agents, binders,disintegrants, coloring, diluents, exipients, extenders, glidants,solubilizers, stabilizers, tonicity agents, vehicles, viscosity agents,flavoring agents, emulsions, such as oil/water emulsions, emulsifyingand suspending agents, such as acacia, agar, alginic acid, sodiumalginate, bentonite, carbomer, carrageenan, carboxymethylcellulose,cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, methylcellulose, octoxynol 9,oleyl alcohol, povidone, propylene glycol monostearate, sodium laurylsulfate, sorbitan esters, stearyl alcohol, tragacanth, xanthan gum, andderivatives thereof, solvents, and miscellaneous ingredients such ascrystalline cellulose, microcrystalline cellulose, citric acid, dextrin,dextrose, liquid glucose, lactic acid, lactose, magnesium chloride,potassium metaphosphate, starch, among others. Such carriers and/oradditives can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Stabilizing agents suchas lipids, nuclease inhibitors, polymers, and chelating agents canpreserve the compositions from degradation within the body.

Colloidal dispersion systems that can be used for delivery of virusesinclude macromolecule complexes, nanocapsules, microspheres, beads andlipid-based systems including oil-in-water emulsions (mixed), micelles,liposomes and lipoplexes. An exemplary colloidal system is a liposome.Organ-specific or cell-specific liposomes can be used in order toachieve delivery only to the desired tissue. The targeting of liposomescan be carried out by the person skilled in the art by applying commonlyknown methods. This targeting includes passive targeting (utilizing thenatural tendency of the liposomes to distribute to cells of the RES inorgans which contain sinusoidal capillaries) or active targeting (forexample, by coupling the liposome to a specific ligand, for example, anantibody, a receptor, sugar, glycolipid and protein by methods know tothose of skill in the art). In the present methods, monoclonalantibodies can be used to target liposomes to specific tissues, forexample, tumor tissue, via specific cell-surface ligands.

2. Host Cells

Also provided herein are host cells that contain a virus providedherein, such as a modified vaccinia virus. Such cells can be group of asingle type of cells or a mixture of different types of cells. Hostcells can include cultured cell lines, primary cells, and proliferativecells. These host cells can include any of a variety of animal cells,such as mammalian, avian and insect cells and tissues that aresusceptible to the virus, such as vaccinia virus, infection, includingchicken embryo, rabbit, hamster, and monkey kidney cells. Suitable hostcells include but are not limited to hematopoietic cells (totipotent,stem cells, leukocytes, lymphocytes, monocytes, macrophages, APC,dendritic cells, non-human cells and the like), pulmonary cells,tracheal cells, hepatic cells, epithelial cells, endothelial cells,muscle cells (e.g., skeletal muscle, cardiac muscle or smooth muscle),fibroblasts, and cell lines including, for example, CV-1, BSC40, Vero,BSC40 and BSC-1, and human HeLa cells. Methods for transforming thesehost cells, phenotypically selecting transformants, and other suchmethods are known in the art.

3. Combinations

Provided are combinations of the viruses provided herein and a secondagent, such as a second virus or other therapeutic or diagnostic agent.A combination can include any virus or reagent for effecting attenuationthereof in accord with the methods provided herein. Combinations caninclude a virus provided herein with one or more additional viruses.Combinations of the viruses provided can also contain pharmaceuticalcompositions containing the viruses or host cells containing the virusesas described herein.

In one embodiment, the virus in a combination is an attenuated virus,such as for example, an attenuated vaccinia virus. Exemplary attenuatedviruses include vaccinia viruses provided herein, such as, but notlimited to, for example, vaccinia viruses described in the Examples:GLV-1h86, GLV-1j87, GLV-1j88, GLV-1j89, GLV-1h90, GLV-1h91, GLV-1h92,GLV-1h96, GLV-1h97, GLV-1h98, GLV-1h104, GLV-1h105, GLV-1h106,GLV-1h107, GLV-1h108 and GLV-1h109.

Combinations provided herein can contain a virus and a therapeuticcompound. Therapeutic compounds for the compositions provided herein canbe, for example, an anti-cancer or chemotherapeutic compound. Exemplarytherapeutic compounds include, for example, cytokines, growth factors,photosensitizing agents, radionuclides, toxins, siRNA molecules,enzyme/pro-drug pairs, anti-metabolites, signaling modulators,anti-cancer antibiotics, anti-cancer antibodies, angiogenesisinhibitors, chemotherapeutic compounds, or a combination thereof.Viruses provided herein can be combined with an anti-cancer compound,such as a platinum coordination complex. Exemplary platinum coordinationcomplexes include, for example, cisplatin, carboplatin, oxaliplatin,DWA2114R, NK121, IS 3 295, and 254-S. Additional exemplary therapeuticcompounds for the use in pharmaceutical composition combinations can befound elsewhere herein (see e.g., Section I. THERAPEUTIC METHODS forexemplary cytokines, growth factors, photosensitizing agents,radionuclides, toxins, siRNA molecules, enzyme/pro-drug pairs,anti-metabolites, signaling modulators, anti-cancer antibiotics,anti-cancer antibodies, angiogenesis inhibitors, and chemotherapeuticcompounds). Exemplary chemotherapeutic agents include methotrexate,vincristine, adriamycin, non-sugar containing chloroethylnitrosoureas,5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol,fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan,MM1270, BAY 12-9566, RAS farnesyl transferase inhibitor, farnesyltransferase inhibitor, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec,CI-994, TNP-470, Hycamtin/Topotecan, PKC412, Valspodar/PSC833,Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070, BCH-4556,CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101, IS1641, ODN698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805,DX8951f, Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin,Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomaldoxorubicin, Yewtaxan/Placlitaxel, Taxol/Paclitaxel,Xeload/Capecitabine, Furtulon/Doxifluridine, Cyclopax/oral paclitaxel,Oral Taxoid, SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358(774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, BMS-182751/oralplatinum, UFT (Tegafur/Uracil), Ergamisol/Levamisole,Eniluracil/776C85/5FU enhancer, Campto/Levamisole, Camptosar/Irinotecan,Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel,Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin,Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomaldoxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine seeds,CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide,Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin,Plantinol/cisplatin, Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel,prodrug of guanine arabinoside, Taxane Analog, nitrosoureas, alkylatingagents such as melphelan and cyclophosphamide, Aminoglutethimide,Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl,Dactinomycin, Daunorubicin HCl, Estramustine phosphate sodium, Etoposide(VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolideacetate (LHRH-releasing factor analogue), Lomustine (CCNU),Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna, Mitotane(o.p′-DDD), Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl,Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastinesulfate, Amsacrine (m-AMSA), Azacitidine, Erythropoietin,Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methylglyoxal bis-guanylhydrazone; MGBG), Pentostatin (2′deoxycoformycin),Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate.

In a further embodiment, the combination can include additionaltherapeutic compounds such as, for example, compounds that aresubstrates for enzymes encoded and expressed by the virus, or othertherapeutic compounds provided herein or known in the art to act inconcert with a virus. For example, the virus can express an enzyme thatconverts a prodrug into an active chemotherapy drug for killing thecancer cell. Hence, combinations provided herein can contain therapeuticcompounds, such as prodrugs. An exemplary virus/therapeutic compoundcombination can include a virus encoding Herpes simplex virus thymidinekinase with the prodrug gancyclovir. Additional exemplaryenzyme/pro-drug pairs, for the use in combinations provided include, butare not limited to, varicella zoster thymidine kinase/gancyclovir,cytosine deaminase/5-fluorouracil, purine nucleosidephosphorylase/6-methylpurine deoxyriboside, betalactamase/cephalosporin-doxorubicin, carboxypeptidaseG2/4-[(2-chloroethyl) (2-mesuloxyethyl)amino]benzoyl-L-glutamic acid,cytochrome P450/acetominophen, horseradish peroxidase/indole-3-aceticacid, nitroreductase/CB1954, rabbitcarboxylesterase/7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycampotothecin,mushroomtyrosinase/bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28,beta galactosidase/1-chloromethyl-5-hydroxy-1,2-dihyro-3H-benz[e]indole,beta glucuronidase/epirubicin-glucoronide, thymidinephosphorylase/5′-deoxy-5-fluorouridine, deoxycytidine kinase/cytosinearabinoside, beta-lactamase and linamerase/linamarin. Additionalexemplary prodrugs, for the use in combinations can also be foundelsewhere herein (see e.g., Section I. THERAPEUTIC METHODS). Any of avariety of known combinations provided herein or otherwise known in theart can be included in the combinations provided herein.

In a further embodiment, combinations can include compounds that cankill or inhibit viral growth or toxicity. Combinations provided hereincan contain antibiotic, antifungal, anti-parasitic or antiviralcompounds for treatment of infections. Exemplary antibiotics which canbe included in a combination with a virus provided herein include, butare not limited to, ceftazidime, cefepime, imipenem, aminoglycoside,vancomycin, and antipseudomonal β-lactam. Exemplary antifungal agentswhich can be included in a combination with a virus provided hereininclude, but are not limited to, amphotericin B, dapsone, fluconazole,flucytosine, griseofluvin, intraconazole, ketoconazole, miconazole,clotrimazole, nystatin, and combinations thereof. Exemplary antiviralagents can be included in a combination with a virus provided hereininclude, but are not limited to, cidofovir, alkoxyalkyl esters ofcidofovir (CDV), cyclic CDV, and (S)-9-(3-hydroxy-2phosphonylmethoxypropyl)adenine, 5-(Dimethoxymethyl)-2′-deoxyuridine,isatin-beta-thiosemicarbazone, N-methanocarbathymidine, brivudin,7-deazaneplanocin A, ST-246, Gleevec,2′-beta-fluoro-2′,3′-dideoxyadenosine, indinavir, nelfinavir, ritonavir,nevirapine, AZT, ddI, ddC, and combinations thereof. Typically,combinations with an antiviral agent contain an antiviral agent known tobe effective against the virus of the combination. For example,combinations can contain a vaccinia virus with an antiviral compound,such as cidofovir, alkoxyalkyl esters of cidofovir, gancyclovir,acyclovir, ST-246, and Gleevec.

In another embodiment, the combination can further include a detectablecompound. A detectable compound can include a ligand or substrate orother compound that can interact with and/or bind specifically to avirally expressed protein or RNA molecule, and can provide a detectablesignal, such as a signal detectable by tomographic, spectroscopic,magnetic resonance, or other known techniques. Exemplary detectablecompounds can be, or can contain, an imaging agent such as a magneticresonance, ultrasound or tomographic imaging agent, including aradionuclide. The detectable compound can include any of a variety ofcompounds as provided elsewhere herein or are otherwise known in theart. Typically, the detectable compound included with a virus in thecombinations provided herein will be a compound that is a substrate, aligand, or can otherwise specifically interact with, a protein or RNAencoded by the virus; in some examples, the protein or RNA is anexogenous protein or RNA. Exemplary viruses/detectable compounds includea virus encoding luciferase/luciferin,β-galactosidase/(4,7,10-tri(aceticacid)-1-(2-β-galactopyranosylethoxy)-1,4,7,10-tetraazacyclododecane)gadolinium (Egad), and other combinations known in the art.

In another embodiment, the combination can further include a virus geneexpression modulating compound. Compounds that modulate gene expressionare known in the art, and include, but are not limited to,transcriptional activators, inducers, transcriptional suppressors, RNApolymerase inhibitors, and RNA binding compounds such as siRNA orribozymes. Any of a variety of gene expression modulating compoundsknown in the art can be included in the combinations provided herein.Typically, the gene expression modulating compound included with a virusin the combinations provided herein will be a compound that can bind,inhibit, or react with one or more compounds, active in gene expressionsuch as a transcription factor or RNA of the virus of the combination.An exemplary virus/expression modulator can be a virus encoding achimeric transcription factor complex having a mutant human progesteronereceptor fused to a yeast GAL4 DNA-binding domain an activation domainof the herpes simplex virus protein VP16 and also containing a syntheticpromoter containing a series of GAL4 recognition sequences upstream ofthe adenovirus major late E1B TATA box, where the compound can be RU486(see, e.g., Yu et al., (2002) Mol Genet Genomics 268:169-178). A varietyof other virus/expression modulator combinations known in the art alsocan be included in the combinations provided herein.

In a further embodiment, combination can further contain nanoparticles.Nanoparticles can be designed such that they carry one or moretherapeutic agents provided herein. Additionally, nanoparticles can bedesigned to carry a molecule that targets the nanoparticle to the tumorcells. In one non-limiting example, nanoparticles can be coated with aradionuclide and, optionally, an antibody immunoreactive with atumor-associated antigen.

4. Kits

The viruses, cells, pharmaceutical compositions, or combinationsprovided herein can be packaged as kits. Kits can optionally include oneor more components such as instructions for use, devices, and additionalreagents, and components, such as tubes, containers and syringes forpractice of the methods. Exemplary kits can include the viruses providedherein, and can optionally include instructions for use, a device fordetecting a virus in a subject, a device for administering the virus toa subject, and a device for administering a compound to a subject.

In one example, a kit can contain instructions. Instructions typicallyinclude a tangible expression describing the virus and, optionally,other components included in the kit, and methods for administration,including methods for determining the proper state of the subject, theproper dosage amount, and the proper administration method, foradministering the virus. Instructions can also include guidance formonitoring the subject over the duration of the treatment time.

In another example, a kit can contain a device for detecting a virus ina subject. Devices for detecting a virus in a subject can include a lowlight imaging device for detecting light, for example, emitted fromluciferase, or fluoresced from fluorescent protein, such as a green orred fluorescent protein, a magnetic resonance measuring device such asan MRI or NMR device, a tomographic scanner, such as a PET, CT, CAT,SPECT or other related scanner, an ultrasound device, or other devicethat can be used to detect a protein expressed by the virus within thesubject. Typically, the device of the kit will be able to detect one ormore proteins expressed by the virus of the kit. Any of a variety ofkits containing viruses and detection devices can be included in thekits provided herein, for example, a virus expressing luciferase and alow light imager, or a virus expressing fluorescent protein, such as agreen or red fluorescent protein, and a low light imager.

Kits provided herein also can include a device for administering a virusto a subject. Any of a variety of devices known in the art foradministering medications or vaccines can be included in the kitsprovided herein. Exemplary devices include, but are not limited to, ahypodermic needle, an intravenous needle, a catheter, a needle-lessinjection device, an inhaler, and a liquid dispenser, such as aneyedropper. Typically, the device for administering a virus of the kitwill be compatible with the virus of the kit; for example, a needle-lessinjection device such as a high pressure injection device can beincluded in kits with viruses not damaged by high pressure injection,but is typically not included in kits with viruses damaged by highpressure injection.

Kits provided herein also can include a device for administering acompound to a subject. Any of a variety of devices known in the art foradministering medications to a subject can be included in the kitsprovided herein. Exemplary devices include a hypodermic needle, anintravenous needle, a catheter, a needle-less injection, but are notlimited to, a hypodermic needle, an intravenous needle, a catheter, aneedle-less injection device, an inhaler, and a liquid dispenser such asan eyedropper. Typically the device for administering the compound ofthe kit will be compatible with the desired method of administration ofthe compound. For example, a compound to be delivered subcutaneously canbe included in a kit with a hypodermic needle and syringe.

I. THERAPEUTIC METHODS

Provided herein are therapeutic methods, including methods of treatingand/or preventing immunoprivileged cells or tissue, including cancerouscells, tumors and metastases. Such sites, diseases and disorders includesites of cell proliferation, proliferative conditions, neoplasms,tumors, neoplastic disease, wounds and inflammation. The therapeuticmethods provided herein include, but are not limited to, administering avirus provided herein to a subject containing a tumor and/or metastases.Viruses provided herein include viruses that have been modified usingthe methods provided herein. The administered viruses can posses one ormore characteristics including attenuated pathogenicity, low toxicity,preferential accumulation in tumor, ability to activate an immuneresponse against tumor cells, immunogenicity, replication competence,ability to express exogenous genes, and ability to elicit antibodyproduction against an expressed gene product. The viruses can beadministered for diagnosis and/or therapy of subjects, such as, but notlimited to humans and other mammals, including rodents, dogs, cats,primates, or livestock.

In some embodiments, the viruses can accumulate in tumors or metastases.In some embodiments, the administration of a virus provided hereinresults in a slowing of tumor growth. In other embodiments, theadministration of a virus provided herein results in a decrease in tumorvolume. The therapeutic methods provided herein, however, do not requirethe administered virus to kill tumor cells or decrease the tumor size.Instead, the methods provided herein include administering to a subjecta virus provided herein that can cause or enhance an anti-tumor immuneresponse in the subject. In some embodiments, the viruses providedherein can be administered to a subject without causing viral-induceddisease in the subject. In some embodiments, the viruses can elicit ananti-tumor immune response in the subject, where typically theviral-mediated anti-tumor immune response can develop, for example, overseveral days, a week or more, 10 days or more, two weeks or more, or amonth or more. In some exemplary methods, the virus can be present inthe tumor, and can cause an anti-tumor immune response without the virusitself causing enough tumor cell death to prevent tumor growth. In someembodiments, the tumor is a monotherapeutic tumor or monotherapeuticcancer, where the tumor or cancer does not decrease in volume whentreated with the virus or a therapeutic agent alone.

In some embodiments, provided herein are methods for eliciting orenhancing antibody production against a selected antigen or a selectedantigen type in a subject, where the methods include administering to asubject a virus that can accumulate in a tumor and/or metastasis, andcan cause release of a selected antigen or selected antigen type fromthe tumor, resulting in antibody production against the selected antigenor selected antigen type. Any of a variety of antigens can be targetedin the methods provided herein, including a selected antigen such as anexogenous gene product expressed by the virus, or a selected antigentype such as one or more tumor antigens release from the tumor as aresult of viral infection of the tumor (e.g., by lysis, apoptosis,secretion or other mechanism of causing antigen release from the tumor).

In some embodiments, it can be desirable to maintain release of theselected antigen or selected antigen type over a series of days, forexample, at least a week, at least ten days, at least two weeks or atleast a month. Provided herein are methods for providing a sustainedantigen release within a subject, where the methods includeadministering to a subject a virus that can accumulate in a tumor and/ormetastasis, and can cause sustained release of an antigen, resulting inantibody production against the antigen. The sustained release ofantigen can result in an immune response by the viral-infected host, inwhich the host can develop antibodies against the antigen, and/or thehost can mount an immune response against cells expressing the antigen,including an immune response against tumor cells. Thus, the sustainedrelease of antigen can result in immunization against tumor cells. Insome embodiments, the viral-mediated sustained antigen release-inducedimmune response against tumor cells can result in complete removal orkilling of all tumor cells.

In some embodiments, the therapeutic methods provided herein inhibittumor growth in a subject, where the methods include administering to asubject a virus that can accumulate in a tumor and/or metastasis, andcan cause or enhance an anti-tumor immune response. The anti-tumorimmune response induced as a result of tumor or metastases-accumulatedviruses can result in inhibition of tumor growth.

In some embodiments, the therapeutic methods provided herein inhibitgrowth or formation of a metastasis in a subject, where the methodsinclude administering to a subject a virus provided herein that canaccumulate in a tumor and/or metastasis, and can cause or enhance ananti-tumor immune response. The anti-tumor immune response induced as aresult of tumor or metastasis-accumulated viruses can result ininhibition of metastasis growth or formation.

In other embodiments, the therapeutic methods provided herein decreasethe size of a tumor and/or metastasis in a subject, where the methodsinclude administering to a subject a virus provided herein that canaccumulate in a tumor and/or metastasis, and can cause or enhance ananti-tumor immune response. The anti-tumor immune response induced as aresult of tumor or metastasis-accumulated viruses can result in adecrease in the size of the tumor and/or metastasis.

In some embodiments, the therapeutic methods provided herein eliminate atumor and/or metastasis from a subject, where the methods includeadministering to a subject a virus provided herein that can accumulatein a tumor and/or metastasis, and can cause or enhance an anti-tumorimmune response. The anti-tumor immune response induced as a result oftumor or metastasis-accumulated viruses can result in elimination of thetumor and/or metastasis from the subject.

Methods of reducing or inhibiting tumor growth, inhibiting metastasisgrowth and/or formation, decreasing the size of a tumor or metastasis,eliminating a tumor or metastasis, or other tumor therapeutic methodsprovided herein include causing or enhancing an anti-tumor immuneresponse in the host. The immune response of the host, being anti-tumorin nature, can be mounted against tumors and/or metastases in whichviruses have accumulated, and can also be mounted against tumors and/ormetastases in which viruses have not accumulated, including tumorsand/or metastases that form after administration of the virus to thesubject. Accordingly, a tumor and/or metastasis whose growth orformation is inhibited, or whose size is decreased, or that iseliminated, can be a tumor and/or metastasis in which the viruses haveaccumulated, or also can be a tumor and/or metastasis in which theviruses have not accumulated. Accordingly, provided herein are methodsof reducing or inhibiting tumor growth, inhibiting metastasis growthand/or formation, decreasing the size of a tumor or metastasis,eliminating a tumor or metastasis, or other tumor therapeutic methods,where the method includes administering to a subject a virus providedherein, where the virus accumulates in at least one tumor or metastasisand causes or enhances an anti-tumor immune response in the subject, andthe immune response also is mounted against a tumor and/or metastasis inwhich the virus cell did not accumulate. In another embodiment, methodsare provided for inhibiting or preventing recurrence of a neoplasticdisease or inhibiting or preventing new tumor growth, where the methodsinclude administering to a subject a virus provided herein that canaccumulate in a tumor and/or metastasis, and can cause or enhance ananti-tumor immune response, and the anti-tumor immune response caninhibit or prevent recurrence of a neoplastic disease or inhibit orprevent new tumor growth.

The tumor or neoplastic disease therapeutic methods provided herein,such as methods of reducing or inhibiting tumor growth, inhibitingmetastasis growth and/or formation, decreasing the size of a tumor ormetastasis, eliminating a tumor or metastasis, or other tumortherapeutic methods, also can include administering to a subject a virusprovided herein that can cause tumor cell lysis or tumor cell death.Such a virus can be the same virus as the virus that can cause orenhance an anti-tumor immune response in the subject. Viruses, such asthe viruses provided herein, can cause cell lysis or tumor cell death asa result of expression of an endogenous gene or as a result of anexogenous gene. Endogenous or exogenous genes can cause tumor cell lysisor inhibit cell growth as a result of direct or indirect actions, as isknown in the art, including lytic channel formation or activation of anapoptotic pathway. Gene products, such as exogenous gene products canfunction to activate a prodrug to an active, cytotoxic form, resultingin cell death where such genes are expressed.

Such methods of antigen production or tumor and/or metastasis treatmentcan include administration of a virus provided herein for therapy, suchas for gene therapy, for cancer gene therapy, or for vaccine therapy.Such a virus can be used to stimulate humoral and/or cellular immuneresponse, induce strong cytotoxic T lymphocytes responses in subjectswho can benefit from such responses. For example, the virus can provideprophylactic and therapeutic effects against a tumor infected by thevirus or other infectious diseases, by rejection of cells from tumors orlesions using viruses that express immunoreactive antigens (Earl et al.,Science 234: 728-831 (1986); Lathe et al., Nature (London) 32: 878-880(1987)), cellular tumor-associated antigens (Bemards et al., Proc. Natl.Acad. Sci. USA 84: 6854-6858 (1987); Estin et al., Proc. Natl. Acad.Sci. USA 85: 1052-1056 (1988); Kantor et al., J. Natl. Cancer Inst. 84:1084-1091 (1992); Roth et al., Proc. Natl. Acad. Sci. USA 93: 4781-4786(1996)) and/or cytokines (e.g., IL-2, IL-12), costimulatory molecules(B7-1, B7-2) (Rao et al., J. Immunol. 156: 3357-3365 (1996); Chamberlainet al., Cancer Res. 56: 2832-2836 (1996); Oertli et al., J. Gen. Virol.77: 3121-3125 (1996); Qin and Chatterjee, Human Gene Ther. 7: 1853-1860(1996); McAneny et al., Ann. Surg. Oncol. 3: 495-500 (1996)), or othertherapeutic proteins.

As shown previously, solid tumors can be treated with viruses, such asvaccinia viruses, resulting in an enormous tumor-specific virusreplication, which can lead to tumor protein antigen and viral proteinproduction in the tumors (U.S. Patent Publication No. 2005/0031643).Vaccinia virus administration to mice resulted in lysis of the infectedtumor cells and a resultant release of tumor-cell-specific antigens.Continuous leakage of these antigens into the body led to a very highlevel of antibody titer (in approximately 7-14 days) against tumorproteins, viral proteins, and the virus encoded engineered proteins inthe mice. The newly synthesized anti-tumor antibodies and the enhancedmacrophage, neutrophils count were continuously delivered via thevasculature to the tumor and thereby provided for the recruitment of anactivated immune system against the tumor. The activated immune systemthen eliminated the foreign compounds of the tumor including the viralparticles. This interconnected release of foreign antigens boostedantibody production and continuous response of the antibodies againstthe tumor proteins to function like an autoimmunizing vaccination systeminitiated by vaccinia viral infection and replication, followed by celllysis, protein leakage and enhanced antibody production. Thus, theviruses provided herein and the viruses generated using the methodsprovided herein can be administered in a complete process that can beapplied to all tumor systems with immunoprivileged tumor sites as siteof privileged viral growth, which can lead to tumor elimination by thehost's own immune system.

In other embodiments, methods are provided for immunizing a subject,where the methods include administering to the subject a virus thatexpresses one or more antigens against which antigens the subject willdevelop an immune response. The immunizing antigens can be endogenous tothe virus, such as vaccinia antigens on a vaccinia virus used toimmunize against smallpox, measles, mumps, or the immunizing antigenscan be exogenous antigens expressed by the virus, such as influenza orHIV antigens expressed on a viral capsid surface. In the case ofsmallpox, for example, a tumor specific protein antigen can be carriedby an attenuated vaccinia virus (encoded by the viral genome) for asmallpox vaccine. Thus, the viruses provided herein, including themodified vaccinia viruses can be used as vaccines.

In one embodiment, the tumor treated is a cancer such as pancreaticcancer, non-small cell lung cancer, multiple myeloma, or leukemia,although the cancer is not limited in this respect, and other metastaticdiseases can be treated by the combinations provided herein. Forexample, the tumor treated can be a solid tumor, such as of the lung andbronchus, breast, colon and rectum, kidney, stomach, esophagus, liverand intrahepatic bile duct, urinary bladder, brain and other nervoussystem, head and neck, oral cavity and pharynx, cervix, uterine corpus,thyroid, ovary, testes, prostate, malignant melanoma,cholangiocarcinoma, thymoma, non-melanoma skin cancers, as well ashematologic tumors and/or malignancies, such as childhood leukemia andlymphomas, multiple myeloma, Hodgkin's disease, lymphomas of lymphocyticand cutaneous origin, acute and chronic leukemia such as acutelymphoblastic, acute myelocytic or chronic myelocytic leukemia, plasmacell neoplasm, lymphoid neoplasm and cancers associated with AIDS.Exemplary tumors include, for example, pancreatic tumors, ovariantumors, lung tumors, colon tumors, prostate tumors, cervical tumors andbreast tumors. In one embodiment, the tumor is a carcinoma such as, forexample, an ovarian tumor or a pancreatic tumor.

1. Administration

In performing the therapeutic methods provided herein, a virus can beadministered to a subject, including a subject having a tumor or havingneoplastic cells, or a subject to be immunized. An administered viruscan be a virus provided herein or any other virus generated using themethods provided herein. In some embodiments, the virus administered isa virus containing a characteristic such as attenuated pathogenicity,low toxicity, preferential accumulation in tumor, ability to activate animmune response against tumor cells, high immunogenicity, replicationcompetence, and ability to express exogenous proteins, and combinationsthereof.

a. Steps Prior to Administering the Virus

In some embodiments, one or more steps can be performed prior toadministration of the virus to the subject. Any of a variety ofpreceding steps can be performed, including, but not limited todiagnosing the subject with a condition appropriate for virusadministration, determining the immunocompetence of the subject,immunizing the subject, treating the subject with a chemotherapeuticagent, treating the subject with radiation, or surgically treating thesubject.

For embodiments that include administering a virus to a tumor-bearingsubject for therapeutic purposes, the subject has typically beenpreviously diagnosed with a neoplastic condition. Diagnostic methodsalso can include determining the type of neoplastic condition,determining the stage of the neoplastic conditions, determining the sizeof one or more tumors in the subject, determining the presence orabsence of metastatic or neoplastic cells in the lymph nodes of thesubject, or determining the presence of metastases of the subject. Someembodiments of therapeutic methods for administering a virus to asubject can include a step of determination of the size of the primarytumor or the stage of the neoplastic disease, and if the size of theprimary tumor is equal to or above a threshold volume, or if the stageof the neoplastic disease is at or above a threshold stage, a virus isadministered to the subject. In a similar embodiment, if the size of theprimary tumor is below a threshold volume, or if the stage of theneoplastic disease is at or below a threshold stage, the virus is notyet administered to the subject; such methods can include monitoring thesubject until the tumor size or neoplastic disease stage reaches athreshold amount, and then administering the virus to the subject.Threshold sizes can vary according to several factors, including rate ofgrowth of the tumor, ability of the virus to infect a tumor, andimmunocompetence of the subject. Generally the threshold size will be asize sufficient for a virus to accumulate and replicate in or near thetumor without being completely removed by the host's immune system, andwill typically also be a size sufficient to sustain a virus infectionfor a time long enough for the host to mount an immune response againstthe tumor cells, typically about one week or more, about ten days ormore, or about two weeks or more. Exemplary threshold tumor sizes forviruses, such as vaccinia viruses, are at least about 100 mm³, at leastabout 200 mm³, at least about 300 mm³, at least about 400 mm³, at leastabout 500 mm³, at least about 750 mm³, at least about 1000 mm³, or atleast about 1500 mm³. Threshold neoplastic disease stages also can varyaccording to several factors, including specific requirement for staginga particular neoplastic disease, aggressiveness of growth of theneoplastic disease, ability of the virus to infect a tumor ormetastasis, and immunocompetence of the subject. Generally the thresholdstage will be a stage sufficient for a virus to accumulate and replicatein a tumor or metastasis without being completely removed by the host'simmune system, and will typically also be a size sufficient to sustain avirus infection for a time long enough for the host to mount an immuneresponse against the neoplastic cells, typically about one week or more,about ten days or more, or about two weeks or more. Exemplary thresholdstages are any stage beyond the lowest stage (e.g., Stage I orequivalent), or any stage where the primary tumor is larger than athreshold size, or any stage where metastatic cells are detected.

In other embodiments, prior to administering to the subject a virus, theimmunocompetence of the subject can be determined. The methods ofadministering a virus to a subject provided herein can include causingor enhancing an immune response in a subject. Accordingly, prior toadministering a virus to a subject, the ability of a subject to mount animmune response can be determined. Any of a variety of tests ofimmunocompetence known in the art can be performed in the methodsprovided herein. Exemplary immunocompetence tests can examine ABOhemagglutination titers (IgM), leukocyte adhesion deficiency (LAD),granulocyte function (NBT), T and B cell quantitation, tetanus antibodytiters, salivary IgA, skin test, tonsil test, complement C3 levels, andfactor B levels, and lymphocyte count. One skilled in the art candetermine the desirability to administer a virus to a subject accordingto the level of immunocompetence of the subject, according to theimmunogenicity of the virus, and, optionally, according to theimmunogenicity of the neoplastic disease to be treated. Typically, asubject can be considered immunocompetent if the skilled artisan candetermine that the subject is sufficiently competent to mount an immuneresponse against the virus.

In some embodiments, the subject can be immunized prior to administeringto the subject a virus according to the methods provided herein.Immunization can serve to increase the ability of a subject to mount animmune response against the virus, or increase the speed at which thesubject can mount an immune response against a virus. Immunization alsocan serve to decrease the risk to the subject of pathogenicity of thevirus. In some embodiments, the immunization can be performed with animmunization virus that is similar to the therapeutic virus to beadministered. For example, the immunization virus can be areplication-incompetent variant of the therapeutic virus. In otherembodiments, the immunization material can be digests of the therapeuticvirus to be administered. Any of a variety of methods for immunizing asubject against a known virus are known in the art and can be usedherein. In one example, vaccinia viruses treated with, for example, 1microgram of psoralen and ultraviolet light at 365 nm for 4 minutes, canbe rendered replication incompetent. In another embodiment, the viruscan be selected as the same or similar to a virus against which thesubject has been previously immunized, e.g., in a childhood vaccination.

In another embodiment, the subject can have administered thereto a viruswithout any previous steps of cancer treatment such as chemotherapy,radiation therapy or surgical removal of a tumor and/or metastases. Themethods provided herein take advantage of the ability of the viruses toenter or localize near a tumor, where the tumor cells can be protectedfrom the subject's immune system; the viruses can then proliferate insuch an immunoprotected region and can also cause the release, typicallya sustained release, of tumor antigens from the tumor to a location inwhich the subject's immune system can recognize the tumor antigens andmount an immune response. In such methods, existence of a tumor ofsufficient size or sufficiently developed immunoprotected state can beadvantageous for successful administration of the virus to the tumor,and for sufficient tumor antigen production. If a tumor is surgicallyremoved, the viruses may not be able to localize to other neoplasticcells (e.g., small metastases) because such cells have not yet havematured sufficiently to create an immunoprotective environment in whichthe viruses can survive and proliferate, or even if the viruses canlocalize to neoplastic cells, the number of cells or size of the masscan be too small for the viruses to cause a sustained release of tumorantigens in order for the host to mount an anti-tumor immune response.Thus, for example, provided herein are methods of treating a tumor orneoplastic disease in which viruses are administered to a subject with atumor or neoplastic disease without removing the primary tumor, or to asubject with a tumor or neoplastic disease in which at least some tumorsor neoplastic cells are intentionally permitted to remain in thesubject. In other typical cancer treatment methods such as chemotherapyor radiation therapy, such methods typically have a side effect ofweakening the subject's immune system. This treatment of a subject bychemotherapy or radiation therapy can reduce the subject's ability tomount an anti-tumor immune response. Thus, for example, provided hereinare methods of treating a tumor or neoplastic disease in which virusesare administered to a subject with a tumor or neoplastic disease withouttreating the subject with an immune system-weakening therapy, such aschemotherapy or radiation therapy.

In an alternative embodiment, prior to administration of a virus to thesubject, the subject can be treated in one or more cancer treatmentsteps that do not remove the primary tumor or that do not weaken theimmune system of the subject. A variety of more sophisticated cancertreatment methods are being developed in which the tumor can be treatedwithout surgical removal or immune-system weakening therapy. Exemplarymethods include administering a compound that decreases the rate ofproliferation of the tumor or neoplastic cells without weakening theimmune system (e.g., by administering tumor suppressor compounds or byadministering tumor cell-specific compounds) or administering anangiogenesis-inhibiting compound. Thus, combined methods that includeadministering a virus to a subject can further improve cancer therapy.Thus, provided herein are methods of administering a virus to a subject,along with prior to or subsequent to, for example, administering acompound that slows tumor growth without weakening the subject's immunesystem or a compound that inhibits vascularization of the tumor.

b. Mode of administration

1. Any mode of administration of a virus to a subject can be used,provided the mode of administration permits the virus to enter a tumoror metastasis. Modes of administration can include, but are not limitedto, systemic, intravenous, intraperitoneal, subcutaneous, intramuscular,transdermal, intradermal, intra-arterial (e.g., hepatic arteryinfusion), intravesicular perfusion, intrapleural, intraarticular,topical, intratumoral, intralesional, multipuncture (e.g., as used withsmallpox vaccines), inhalation, percutaneous, subcutaneous, intranasal,intratracheal, oral, intracavity (e.g., administering to the bladder viaa catheter, administering to the gut by suppository or enema), vaginal,rectal, intracranial, intraprostatic, intravitreal, aural, or ocularadministration.

One skilled in the art can select any mode of administration compatiblewith the subject and the virus, and that also is likely to result in thevirus reaching tumors and/or metastases. The route of administration canbe selected by one skilled in the art according to any of a variety offactors, including the nature of the disease, the kind of tumor, and theparticular virus contained in the pharmaceutical composition.Administration to the target site can be performed, for example, byballistic delivery, as a colloidal dispersion system, or systemicadministration can be performed by injection into an artery.

c. Dosages

The dosage regimen can be any of a variety of methods and amounts, andcan be determined by one skilled in the art according to known clinicalfactors. As is known in the medical arts, dosages for any one patientcan depend on many factors, including the subject's species, size, bodysurface area, age, sex, immunocompetence, and general health, theparticular virus to be administered, duration and route ofadministration, the kind and stage of the disease, for example, tumorsize, and other treatments or compounds, such as chemotherapeutic drugs,being administered concurrently. In addition to the above factors, suchlevels can be affected by the infectivity of the virus, and the natureof the virus, as can be determined by one skilled in the art. In thepresent methods, appropriate minimum dosage levels of viruses can belevels sufficient for the virus to survive, grow and replicate in atumor or metastasis. Exemplary minimum levels for administering a virusto a 65 kg human can include at least about 1×10⁵ plaque forming units(PFU), at least about 5×10⁵ PFU, at least about 1×10⁶ PFU, at leastabout 5×10⁶ PFU, at least about 1×10⁷ PFU, at least about 1×10⁸ PFU, atleast about 1×10⁹ PFU, or at least about 1×10¹⁰ PFU. In the presentmethods, appropriate maximum dosage levels of viruses can be levels thatare not toxic to the host, levels that do not cause splenomegaly of 3times or more, levels that do not result in colonies or plaques innormal tissues or organs after about 1 day or after about 3 days orafter about 7 days. Exemplary maximum levels for administering a virusto a 65 kg human can include no more than about 1×10¹¹ PFU, no more thanabout 5×10¹⁰ PFU, no more than about 1×10¹⁰ PFU, no more than about5×10⁹ PFU, no more than about 1×10⁹ PFU, or no more than about 1×10⁸PFU.

For combination therapies with chemotherapeutic compounds, dosages forthe administration of such compounds are known in the art or can bedetermined by one skilled in the art according to known clinical factors(e.g., subject's species, size, body surface area, age, sex,immunocompetence, and general health, duration and route ofadministration, the kind and stage of the disease, for example, tumorsize, and other viruses, treatments, or compounds, such as otherchemotherapeutic drugs, being administered concurrently). In addition tothe above factors, such levels can be affected by the infectivity of thevirus, and the nature of the virus, as can be determined by one skilledin the art. For example, Cisplatin (also called cis-platinum, platinol;cis-diamminedichloroplatinum; and cDDP) is representative of a broadclass of water-soluble, platinum coordination compounds frequentlyemployed in the therapy of testicular cancer, ovarian tumors, and avariety of other cancers. (See, e.g., Blumenreich et al. Cancer 55(5):1118-1122 (1985); Forastiere et al. J. Clin. Oncol. 19(4): 1088-1095(2001)). Methods of employing cisplatin clinically are well known in theart. For example, cisplatin has been administered in a single day over asix hour period, once per month, by slow intravenous infusion. Forlocalized lesions, cisplatin can be administered by local injection.Intraperitoneal infusion can also be employed. Cisplatin can beadministered in doses as low as 10 mg/m² per treatment if part of amulti-drug regimen, or if the patient has an adverse reaction to higherdosing. In general, a clinical dose is from about 30 to about 120 or 150mg/m² per treatment.

Typically, platinum-containing chemotherapeutic agents are administeredparenterally, for example by slow intravenous infusion, or by localinjection, as discussed above. The effects of intralesional(intra-tumoral) and IP administration of cisplatin is described in(Nagase et al. Cancer Treat. Rep. 71(9): 825-829 (1987); and Theon etal. J. Am. Vet. Med. Assoc. 202(2): 261-7. (1993)).

In one exemplary embodiment, the mutant vaccinia virus is administeredonce or 2-4 times with 0-60 days apart, followed by 1-30 days where noanti-cancer treatment, then cisplatin is administered daily for 1-5days, followed by 1-30 days where no anti-cancer treatment isadministered. Each component of the therapy, virus or cisplatintreatment, or the virus and cisplatin combination therapy can berepeated. In another exemplary embodiment, cisplatin is administereddaily for 1 to 5 days, followed by 1-10 days where no anti-cancertreatment is administered, then the mutant vaccinia virus isadministered once or 2-4 times with 0-60 days apart. Such treatmentscheme can be repeated. In another exemplary embodiment, cisplatin isadministered daily for 1 to 5 days, followed by 1-10 days where noanti-cancer treatment is administered, then the mutant vaccinia virus isadministered once or 2-4 times with 0-60 days apart. This is followed by5-60 days where no anti-cancer treatment is administered, then cisplatinis administered again for 1-5 days. Such treatment scheme can berepeated.

Gemcitabine (GEMZAR®) is another compound employed in the therapy ofbreast cancer, non-small cell lung cancer, and pancreatic cancer.Gemcitabine is a nucleoside analogue that exhibits antitumor activity.Methods of employing gemcitabine clinically are well known in the art.For example, gemcitabine has been administered by intravenous infusionat a dose of 1000 mg/m² over 30 minutes once weekly for up to 7 weeks(or until toxicity necessitates reducing or holding a dose), followed bya week of rest from treatment of pancreatic cancer. Subsequent cyclescan consist of infusions once weekly for 3 consecutive weeks out ofevery 4 weeks. Gemcitabine has also been employed in combination withcisplatin in cancer therapy.

In one exemplary embodiment, the mutant vaccinia virus is administeredonce or 2-4 times with 0-60 days apart, followed by 1-30 days where noanti-cancer treatment is administered, then gemcitabine is administered1-7 times with 0-30 days apart, followed by 1-30 days where noanti-cancer treatment is administered. Such treatment scheme can berepeated. In another exemplary embodiment, gemcitabine is administered1-7 times with 0-30 days apart, followed by 1-10 days where noanti-cancer treatment is administered, then the mutant vaccinia virus isadministered once or 2-4 times with 0-60 days apart. This is followed by5-60 days where no anti-cancer treatment is administered. Such treatmentscheme can be repeated. In another exemplary embodiment, gemcitabine isadministered 1-7 times with 0-30 days apart, followed by 1-10 days whereno anti-cancer treatment is administered, then the mutant vaccinia virusis administered once or 2-4 times with 0-60 days apart. This is followedby 5-60 days where no anti-cancer treatment is administered, thengemcitabine is administered again for 1-7 times with 0-30 days apart.Such treatment scheme can be repeated.

As will be understood by one of skill in the art, the optimal treatmentregimen will vary and it is within the scope of the treatment methods toevaluate the status of the disease under treatment and the generalhealth of the patient prior to, and following one or more cycles ofcombination therapy in order to determine the optimal therapeuticcombination.

d. Number of Administrations

The methods provided herein can include a single administration of avirus to a subject or multiple administrations of a virus to a subject.In some embodiments, a single administration is sufficient to establisha virus in a tumor, where the virus can proliferate and can cause orenhance an anti-tumor response in the subject; such methods do notrequire additional administrations of a virus in order to cause orenhance an anti-tumor response in a subject, which can result, forexample in inhibition of tumor growth, inhibition of metastasis growthor formation, reduction in tumor or size, elimination of a tumor ormetastasis, inhibition or prevention of recurrence of a neoplasticdisease or new tumor formation, or other cancer therapeutic effects. Inother embodiments, a virus can be administered on different occasions,separated in time typically by at least one day. Separateadministrations can increase the likelihood of delivering a virus to atumor or metastasis, where a previous administration has beenineffective in delivering a virus to a tumor or metastasis. Separateadministrations can increase the locations on a tumor or metastasiswhere virus proliferation can occur or can otherwise increase the titerof virus accumulated in the tumor, which can increase the scale ofrelease of antigens or other compounds from the tumor in eliciting orenhancing a host's anti-tumor immune response, and also can, optionally,increase the level of virus-based tumor lysis or tumor cell death.Separate administrations of a virus can further extend a subject'simmune response against viral antigens, which can extend the host'simmune response to tumors or metastases in which viruses haveaccumulated, and can increase the likelihood of a host mounting ananti-tumor immune response.

When separate administrations are performed, each administration can bea dosage amount that is the same or different relative to otheradministration dosage amounts. In one embodiment, all administrationdosage amounts are the same. In other embodiments, a first dosage amountcan be a larger dosage amount than one or more subsequent dosageamounts, for example, at least 10× larger, at least 100× larger, or atleast 1000× larger than subsequent dosage amounts. In one example of amethod of separate administrations in which the first dosage amount isgreater than one or more subsequent dosage amounts, all subsequentdosage amounts can be the same, smaller amount relative to the firstadministration.

Separate administrations can include any number of two or moreadministrations, including two, three, four, five or sixadministrations. One skilled in the art can readily determine the numberof administrations to perform or the desirability of performing one ormore additional administrations according to methods known in the artfor monitoring therapeutic methods and other monitoring methods providedherein. Accordingly, the methods provided herein include methods ofproviding to the subject one or more administrations of a virus, wherethe number of administrations can be determined by monitoring thesubject, and, based on the results of the monitoring, determiningwhether or not to provide one or more additional administrations.Deciding on whether or not to provide one or more additionaladministrations can be based on a variety of monitoring results,including, but not limited to, indication of tumor growth or inhibitionof tumor growth, appearance of new metastases or inhibition ofmetastasis, the subject's anti-virus antibody titer, the subject'santi-tumor antibody titer, the overall health of the subject, the weightof the subject, the presence of virus solely in tumor and/or metastases,the presence of virus in normal tissues or organs.

The time period between administrations can be any of a variety of timeperiods. The time period between administrations can be a function ofany of a variety of factors, including monitoring steps, as described inrelation to the number of administrations, the time period for a subjectto mount an immune response, the time period for a subject to clear thevirus from normal tissue, or the time period for virus proliferation inthe tumor or metastasis. In one example, the time period can be afunction of the time period for a subject to mount an immune response;for example, the time period can be more than the time period for asubject to mount an immune response, such as more than about one week,more than about ten days, more than about two weeks, or more than abouta month; in another example, the time period can be less than the timeperiod for a subject to mount an immune response, such as less thanabout one week, less than about ten days, less than about two weeks, orless than about a month. In another example, the time period can be afunction of the time period for a subject to clear the virus from normaltissue; for example, the time period can be more than the time periodfor a subject to clear the virus from normal tissue, such as more thanabout a day, more than about two days, more than about three days, morethan about five days, or more than about a week. In another example, thetime period can be a function of the time period for virus proliferationin the tumor or metastasis; for example, the time period can be morethan the amount of time for a detectable signal to arise in a tumor ormetastasis after administration of a virus expressing a detectablemarker, such as about 3 days, about 5 days, about a week, about tendays, about two weeks, or about a month.

e. Co-administrations

Also provided are methods in which an additional therapeutic substance,such as a different therapeutic virus or a therapeutic compound isadministered. These can be administered simultaneously, sequentially orintermittently with the first virus. The additional therapeuticsubstance can interact with the virus or a gene product thereof, or theadditional therapeutic substance can act independently of the virus.

Combination therapy treatment has advantages in that: 1) it avoidssingle agent resistance; 2) in a heterogeneous tumor population, it cankill cells by different mechanisms; and 3) by selecting drugs withnon-overlapping toxicities, each agent can be used at full dose toelicit maximal efficacy and synergistic effect. Combination therapy canbe done by combining a diagnostic/therapeutic virus with one or more ofthe following anti-cancer agents: chemotherapeutic agents, therapeuticantibodies, siRNAs, toxins, enzyme-prodrug pairs, or radiation.

i. Administering a Plurality of Viruses

Methods are provided for administering to a subject two or more viruses.Administration can be effected simultaneously, sequentially orintermittently. The plurality of viruses can be administered as a singlecomposition or as two or more compositions. The two or more viruses caninclude at least two viruses. In a particular embodiment, where thereare two viruses, both viruses are vaccinia viruses. In anotherembodiment, one viruses is a vaccinia virus and the second viruses isany one of an adenovirus, an adeno-associated virus, a retrovirus, aherpes simplex virus, a reovirus, a mumps virus, a foamy virus, aninfluenza virus, a myxoma virus, a vesicular stomatitis virus, or anyother virus described herein or known in the art. Viruses can be chosenbased on the pathway on which they act. For example, a virus thattargets an activated Ras pathway can be combined with a virus thattargets tumor cells defective in p53 expression.

The plurality of viruses can be provided as combinations of compositionscontaining and/or as kits that include the viruses packaged foradministration and optionally including instructions therefore. Thecompositions can contain the viruses formulated for single dosageadministration (i.e., for direct administration) and can requiredilution or other additions.

In one embodiment, at least one of the viruses is a modified virus suchas those provided herein, having a characteristic such as lowpathogenicity, low toxicity, preferential accumulation in tumor, abilityto activate an immune response against tumor cells, immunogenic,replication competent, ability to express exogenous proteins, andcombinations thereof. The viruses can be administered at approximatelythe same time, or can be administered at different times. The virusescan be administered in the same composition or in the sameadministration method, or can be administered in separate composition orby different administration methods.

The time period between administrations can be any time period thatachieves the desired effects, as can be determined by one skilled in theart. Selection of a time period between administrations of differentviruses can be determined according to parameters similar to those forselecting the time period between administrations of the same virus,including results from monitoring steps, the time period for a subjectto mount an immune response, the time period for a subject to clearvirus from normal tissue, or the time period for virus proliferation inthe tumor or metastasis. In one example, the time period can be afunction of the time period for a subject to mount an immune response;for example, the time period can be more than the time period for asubject to mount an immune response, such as more than about one week,more than about ten days, more than about two weeks, or more than abouta month; in another example, the time period can be less than the timeperiod for a subject to mount an immune response, such as less thanabout one week, less than about ten days, less than about two weeks, orless than about a month. In another example, the time period can be afunction of the time period for a subject to clear the virus from normaltissue; for example, the time period can be more than the time periodfor a subject to clear the virus from normal tissue, such as more thanabout a day, more than about two days, more than about three days, morethan about five days, or more than about a week. In another example, thetime period can be a function of the time period for virus proliferationin the tumor or metastasis; for example, the time period can be morethan the amount of time for a detectable signal to arise in a tumor ormetastasis after administration of a virus expressing a detectablemarker, such as about 3 days, about 5 days, about a week, about tendays, about two weeks, or about a month.

ii. Therapeutic Compounds

Any therapeutic or anti-cancer agent can be used as the second,therapeutic or anti-cancer agent in the combined cancer treatmentmethods provided herein. The methods can include administering one ormore therapeutic compounds to the subject in addition to administering avirus or plurality thereof to a subject. Therapeutic compounds can actindependently, or in conjunction with the virus, for tumor therapeuticeffects.

Therapeutic compounds that can act independently include any of avariety of known chemotherapeutic compounds that can inhibit tumorgrowth, inhibit metastasis growth and/or formation, decrease the size ofa tumor or metastasis, eliminate a tumor or metastasis, without reducingthe ability of a virus to accumulate in a tumor, replicate in the tumor,and cause or enhance an anti-tumor immune response in the subject.

Therapeutic compounds that act in conjunction with the viruses include,for example, compounds that alter the expression of the viruses orcompounds that can interact with a virally-expressed gene, or compoundsthat can inhibit virus proliferation, including compounds toxic to thevirus. Therapeutic compounds that can act in conjunction with the virusinclude, for example, therapeutic compounds that increase theproliferation, toxicity, tumor cell killing, or immune responseeliciting properties of a virus, and also can include, for example,therapeutic compounds that decrease the proliferation, toxicity, or cellkilling properties of a virus. Optionally, the therapeutic agent canexhibit or manifest additional properties, such as, properties thatpermit its use as an imaging agent, as described elsewhere herein.

Therapeutic compounds also include, but are not limited to,chemotherapeutic agents, nanoparticles, radiation therapy, siRNAmolecules, enzyme/pro-drug pairs, photosensitizing agents, toxins,microwaves, a radionuclide, an angiogenesis inhibitor, a mitosisinhibitor protein (e.g., cdc6), an antitumor oligopeptide (e.g.,antimitotic oligopeptides, high affinity tumor-selective bindingpeptides), a signaling modulator, anti-cancer antibiotics, or acombination thereof.

Exemplary photosensitizing agents include, but are not limited to, forexample, indocyanine green, toluidine blue, aminolevulinic acid,texaphyrins, benzoporphyrins, phenothiazines, phthalocyanines,porphyrins such as sodium porfimer, chlorins such astetra(m-hydroxyphenyl)chlorin or tin(IV) chlorin e6, purpurins such astin ethyl etiopurpurin, purpurinimides, bacteriochlorins, pheophorbides,pyropheophorbides or cationic dyes. In one embodiment, a vaccinia virus,such as a vaccinia virus provided herein, is administered to a subjecthaving a tumor, cancer or metastasis in combination with aphotosensitizing agent.

Radionuclides, which depending up the radionuclide, amount andapplication can be used for diagnosis and/or for treatment. Theyinclude, but are not limited to, for example, a compound or moleculecontaining ³²Phosphate, ⁶⁰Cobalt, ⁹⁰Yttirum, ⁹⁹Technicium, ¹⁰³Palladium, ¹⁰⁶Ruthenium, ¹¹¹Indium, ¹¹⁷Lutetium, ¹²⁵Iodine, 131Iodine,¹³⁷Cesium, ¹⁵³Samarium, ⁸⁶Rhenium, ¹⁸⁸Rhenium, ¹⁹²Iridium, ¹⁹⁸Gold,²¹¹Astatine, ²¹²Bismuth or ²¹³Bismuth. In one embodiment, a vacciniavirus, such as a vaccinia virus provided herein, is administered to asubject having a tumor, cancer or metastasis in combination with aradionuclide.

Toxins include, but are not limited to, chemotherapeutic compounds suchas, but not limited to, 5-fluorouridine, calicheamicin and maytansine.Signaling modulators include, but are not limited to, for example,inhibitors of macrophage inhibitory factor, toll-like receptor agonistsand stat 3 inhibitors. In one embodiment, a vaccinia virus, such as avaccinia virus provided herein, is administered to a subject having atumor, cancer or metastasis in combination with a toxin or a signalingmodulator.

Combination therapy between chemotherapeutic agents and therapeuticviruses can be effective/curative in situations when single agenttreatment is not effective. Chemotherapeutic compounds include, but arenot limited to, alkylating agents such as thiotepa andcyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime nitrogen mustardssuch as chiorambucil, chlomaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone;mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinicacid; 2-ethylhydrazide; procarbazine; polysaccharide-K; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytosinearabinoside; cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel anddoxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C;mitoxantrone; vincristine; vinorelbine; navelbine; novantrone;teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoic acid; esperamicins; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. Alsoincluded are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone andtoremifene (Fareston); and antiandrogens such as flutamide, nilutamide,bicalutamide, leuprolide and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Such chemotherapeuticcompounds that can be used herein include compounds whose toxicitiespreclude use of the compound in general systemic chemotherapeuticmethods. Chemotherapeutic agents also include new classes of targetedchemotherapeutic agents such as, for example, imatinib (sold by Novartisunder the trade name Gleevec in the United States), gefitinib (developedby Astra Zeneca under the trade name Iressa) and erlotinib. Particularchemotherapeutic agents include, but are not limited to, cisplatin,carboplatin, oxaliplatin, DWA2114R, NK121, IS 3 295, and 254-Svincristine, prednisone, doxorubicin and L-asparaginase;mechoroethamine, vincristine, procarbazine and prednisone (MOPP),cyclophosphamide, vincristine, procarbazine and prednisone (C-MOPP),bleomycin, vinblastine, gemcitabine and 5-fluorouracil. Exemplarychemotherapeutic agents are, for example, cisplatin, carboplatin,oxaliplatin, DWA2114R, NK121, IS 3 295, and 254-S. In a non-limitingembodiment, a vaccinia virus, such as a vaccinia virus provided herein,is administered to a subject having a tumor, cancer or metastasis incombination with a platinum coordination complex, such as cisplatin,carboplatin, oxaliplatin, DWA2114R, NK121, IS 3 295, and 254-S. Tumors,cancers and metastasis can be any of those provided herein, and inparticular, can be a pancreatic tumor, an ovarian tumor, a lung tumor, acolon tumor, a prostate tumor, a cervical tumor or a breast tumor;exemplary tumors are pancreatic and ovarian tumors. Tumors, cancers andmetastasis can be a monotherapy-resistant tumor such as, for example,one that does not respond to therapy with virus alone or anti-canceragent alone, but that does respond to therapy with a combination ofvirus and anti-cancer agent. Typically, a therapeutically effectiveamount of virus is systemically administered to the subject and thevirus localizes and accumulates in the tumor. Subsequent toadministering the virus, the subject is administered a therapeuticallyeffective amount of an anti-cancer agent, such as cisplatin. In oneexample, cisplatin is administered once-daily for five consecutive days.One of skill in the art could determine when to administer theanti-cancer agent subsequent to the virus using, for example, in vivoanimal models. Using the methods provided herein, administration of avirus and anti-cancer agent, such as cisplatin can cause a reduction intumor volume, can cause tumor growth to stop or be delayed or can causethe tumor to be eliminated from the subject. The status of tumors,cancers and metastasis following treatment can be monitored using any ofthe methods provided herein and known in the art.

Exemplary anti-cancer antibiotics include, but are not limited to,anthracyclines such as doxorubicin hydrochloride (adriamycin),idarubicin hydrochloride, daunorubicin hydrochloride, aclarubicinhydrochloride, epirubicin hydrochloride, and purarubicin hydrochloride,pleomycins such as pleomycin and peplomycin sulfate, mitomycins such asmitomycin C, actinomycins such as actinomycin D, zinostatinstimalamer,and polypeptides such as neocarzinostatin. In one embodiment, a vacciniavirus, such as a vaccinia virus provided herein, is administered to asubject having a tumor, cancer or metastasis in combination with ananti-cancer antibiotic.

In one embodiment, nanoparticles can be designed such that they carryone or more therapeutic agents provided herein. Additionally,nanoparticles can be designed to carry a molecule that targets thenanoparticle to the tumor cells. In one non-limiting example,nanoparticles can be coated with a radionuclide and, optionally, anantibody immunoreactive with a tumor-associated antigen. In oneembodiment, a vaccinia virus, such as a vaccinia virus provided herein,is administered to a subject having a tumor, cancer or metastasis incombination with a nanoparticle carrying any of the therapeutic agentsprovided herein.

Radiation therapy has become a foremost choice of treatment for amajority of cancer patients. The wide use of radiation treatment stemsfrom the ability of gamma-irradiation to induce irreversible damage intargeted cells with the preservation of normal tissue function. Ionizingradiation triggers apoptosis, the intrinsic cellular death machinery incancer cells, and the activation of apoptosis seems to be the principalmode by which cancer cells die following exposure to ionizing radiation.In one embodiment, a vaccinia virus, such as a vaccinia virus providedherein, is administered to a subject having a tumor, cancer ormetastasis in combination with radiation therapy.

Thus, provided herein are methods of administering to a subject one ormore therapeutic compounds that can act in conjunction with the virus toincrease the proliferation, toxicity, tumor cell killing, or immuneresponse eliciting properties of a virus. Also provided herein aremethods of administering to a subject one or more therapeutic compoundsthat can act in conjunction with the virus to decrease theproliferation, toxicity, or cell killing properties of a virus.Therapeutic compounds to be administered can be any of those providedherein or in the art.

Therapeutic compounds that can act in conjunction with the virus toincrease the proliferation, toxicity, tumor cell killing, or immuneresponse eliciting properties of a virus are compounds that can altergene expression, where the altered gene expression can result in anincreased killing of tumor cells or an increased anti-tumor immuneresponse in the subject. A gene expression-altering compound can, forexample, cause an increase or decrease in expression of one or moreviral genes, including endogenous viral genes and/or exogenous viralgenes. For example, a gene expression-altering compound can induce orincrease transcription of a gene in a virus such as an exogenous genethat can cause cell lysis or cell death, that can provoke an immuneresponse, that can catalyze conversion of a prodrug-like compound, orthat can inhibit expression of a tumor cell gene. Any of a wide varietyof compounds that can alter gene expression are known in the art,including IPTG and RU486. Exemplary genes whose expression can beup-regulated include proteins and RNA molecules, including toxins,enzymes that can convert a prodrug to an anti-tumor drug, cytokines,transcription regulating proteins, siRNA, and ribozymes. In anotherexample, a gene expression-altering compound can inhibit or decreasetranscription of a gene in a virus such as a heterologous gene that canreduce viral toxicity or reduces viral proliferation. Any of a varietyof compounds that can reduce or inhibit gene expression can be used inthe methods provided herein, including siRNA compounds, transcriptionalinhibitors or inhibitors of transcriptional activators. Exemplary geneswhose expression can be down-regulated include proteins and RNAmolecules, including viral proteins or RNA that suppress lysis,nucleotide synthesis or proliferation, and cellular proteins or RNAmolecules that suppress cell death, immunoreactivity, lysis, or viralreplication.

In another embodiment, therapeutic compounds that can act in conjunctionwith the virus to increase the proliferation, toxicity, tumor cellkilling, or immune response eliciting properties of a virus arecompounds that can interact with a virally expressed gene product, andsuch interaction can result in an increased killing of tumor cells or anincreased anti-tumor immune response in the subject. A therapeuticcompound that can interact with a virally-expressed gene product caninclude, for example a prodrug or other compound that has little or notoxicity or other biological activity in its subject-administered form,but after interaction with a virally expressed gene product, thecompound can develop a property that results in tumor cell death,including but not limited to, cytotoxicity, ability to induce apoptosis,or ability to trigger an immune response. In one non-limiting example,the virus carries an enzyme into the cancer cells. Once the enzyme isintroduced into the cancer cells, an inactive form of a chemotherapydrug (i.e., a prodrug) is administered. When the inactive prodrugreaches the cancer cells, the enzyme converts the prodrug into theactive chemotherapy drug, so that it can kill the cancer cell. Thus, thetreatment is targeted only to cancer cells and does not affect normalcells. The prodrug can be administered concurrently with, orsequentially to, the virus. A variety of prodrug-like substances areknown in the art and an exemplary set of such compounds are disclosedelsewhere herein, where such compounds can include gancyclovir,5-fluorouracil, 6-methylpurine deoxyriboside, cephalosporin-doxorubicin,4-[(2-chloroethyl) (2-mesuloxyethyl)amino]benzoyl-L-glutamic acid,acetaminophen, indole-3-acetic acid, CB1954,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin,bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28,1-chloromethyl-5-hydroxy-1,2-dihyro-3H-benz[e]indole,epirubicin-glucuronide, 5′-deoxy-5-fluorouridine, cytosine arabinoside,linamarin, and a nucleoside analogue (e.g., fluorouridine,fluorodeoxyuridine, fluorouridine arabinoside, cytosine arabinoside,adenine arabinoside, guanine arabinoside, hypoxanthine arabinoside,6-mercaptopurineriboside, theoguanosine riboside, nebularine,5-iodouridine, 5-iododeoxyuridine, 5-bromodeoxyuridine,5-vinyldeoxyuridine, 9-[(2-hydroxy)ethoxy]methylguanine (acyclovir),9-[(2-hydroxy-1-hydroxymethyl)-ethoxy]methylguanine (DHPG), azauridien,azacytidine, azidothymidine, dideoxyadenosine, dideoxycytidine,dideoxyinosine, dideoxyguanosine, dideoxythymidine, 3′-deoxyadenosine,3′-deoxycytidine, 3′-deoxyinosine, 3′-deoxyguanosine,3′-deoxythymidine).

In another embodiment, therapeutic compounds that can act in conjunctionwith the virus to decrease the proliferation, toxicity, or cell killingproperties of a virus are compounds that can inhibit viral replication,inhibit viral toxins, or cause viral death. A therapeutic compound thatcan inhibit viral replication, inhibit viral toxins, or cause viraldeath can generally include a compound that can block one or more stepsin the viral life cycle, including, but not limited to, compounds thatcan inhibit viral DNA replication, viral RNA transcription, viral coatprotein assembly, outer membrane or polysaccharide assembly. Any of avariety of compounds that can block one or more steps in a viral lifecycle are known in the art, including any known antiviral compound(e.g., cidofovir), viral DNA polymerase inhibitors, viral RNA polymeraseinhibitors, inhibitors of proteins that regulate viral DNA replicationor RNA transcription. In another example, a virus can contain a geneencoding a viral life cycle protein, such as DNA polymerase or RNApolymerase that can be inhibited by a compound that is, optionally,non-toxic to the host organism.

In addition to combination therapy between chemotherapeutic agents and avirus provided herein, other more complex combination therapy strategiescould be applied as well. For example, a combination therapy can includechemotherapeutic agents, therapeutic antibodies, and a virus providedherein. Alternatively, another combination therapy can be thecombination of radiation, therapeutic antibodies, and a virus providedherein. Therefore, the concept of combination therapy also can be basedon the application of a virus provided herein virus along with one ormore of the following therapeutic modalities, namely, chemotherapeuticagents, radiation therapy, therapeutic antibodies, hyper- or hypothermiatherapy, siRNA, diagnostic/therapeutic bacteria, diagnostic/therapeuticmammalian cells, immunotherapy, and/or targeted toxins (delivered byantibodies, liposomes and nanoparticles).

Effective delivery of each components of the combination therapy is animportant aspect of the methods provided herein. In accordance with oneaspect, the modes of administration discussed below exploit one of moreof the key features: (i) delivery of a virus provided herein to thetumors by a mode of administration effect to achieve highest titer ofvirus and highest therapeutic effect; (ii) delivery of any othermentioned therapeutic modalities to the tumor by a mode ofadministration to achieve the optimal therapeutic effect. The dosescheme of the combination therapy administered is such that thecombination of the two or more therapeutic modalities is therapeuticallyeffective. Dosages will vary in accordance with such factors as the age,health, sex, size and weight of the patient, the route ofadministration, the toxicity of the drugs, frequency of treatment, andthe relative susceptibilities of the cancer to each of the therapeuticmodalities.

iii. Immunotherapies and Biological Therapies

Therapeutic compounds also include, but are not limited to, compoundsthat exert an immunotherapeutic effect, stimulate the immune system,carry a therapeutic compound, or a combination thereof. Optionally, thetherapeutic agent can exhibit or manifest additional properties, suchas, properties that permit its use as an imaging agent, as describedelsewhere herein. Such therapeutic compounds include, but are notlimited to, anti-cancer antibodies, radiation therapy, siRNA moleculesand compounds that suppress the immune system. Immunotherapy includesfor example, immune-stimulating molecules (protein-based ornon-protein-based), cells and antibodies. Immunotherapy treatments caninclude stimulating immune cells to act more effectively or to make thetumor cells or tumor associated antigens recognizable to the immunesystem (i.e., break tolerance).

Cytokines and growth factors include, but are not limited to,interleukins, such as, for example, interleukin-1, interleukin-2,interleukin-6 and interleukin-12, tumor necrosis factors, such as tumornecrosis factor alpha (TNF-α), interferons such as interferon gamma(IFN-γ), granulocyte macrophage colony stimulating factors (GM-CSF),angiogenins, and tissue factors.

Anti-cancer antibodies include, but are not limited to, Rituximab,ADEPT, Trastuzumab (Herceptin), Tositumomab (Bexxar), Cetuximab(Erbitux), Ibritumomab (Zevalin), Alemtuzumab (Campath-1H), Epratuzumab(Lymphocide), Gemtuzumab ozogamicin (Mylotarg), Bevacimab (Avastin),Tarceva (Erlotinib), SUTENT (sunitinib malate), Panorex (Edrecolomab),RITUXAN (Rituximab), Zevalin (90Y-ibritumomab tiuexetan), Mylotarg(Gemtuzumab Ozogamicin) and Campath (Alemtuzumab).

Thus, provided herein are methods of administering to a subject one ormore therapeutic compounds that can act in conjunction with the virus tostimulate or enhance the immune system, thereby enhancing the effect ofthe virus. Such immunotherapy can be either delivered as a separatetherapeutic modality or could be encoded (if the immunotherapy isprotein-based) by the administered virus.

Biological therapies are treatments that use natural body substances ordrugs made from natural body substances. They can help to treat a cancerand control side effects caused by other cancer treatments such aschemotherapy. Biological therapies are also sometimes called BiologicalResponse Modifiers (BRM's), biologic agents or simply “biologics”because they stimulate the body to respond biologically (or naturally)to cancer. Immunotherapy is treatment using natural substances that thebody uses to fight infection and disease. Because it uses naturalsubstances, immunotherapy is also a biological therapy. There areseveral types of drugs that come under the term biological therapy:these include, for example, monoclonal antibodies (mAbs), cancervaccines, growth factors for blood cells, cancer growth inhibitors,anti-angiogenic factors, interferon alpha, interleukin-2 (IL-2), genetherapy and BCG vaccine for bladder cancer

Monoclonal antibodies (mAbs) are of particular interest for treatingcancer because of the specificity of binding to a unique antigen and theability to produce large quantities in the laboratory for massdistribution. Monoclonal antibodies can be engineered to act in the sameway as immune system proteins: that is, to seek out and kill foreignmatter in your body, such as viruses. Monoclonal antibodies can bedesigned to recognize epitopes on the surface of cancer cells. Theantibodies target specifically bind to the epitopes and either kill thecancer cells or deliver a therapeutic agent to the cancer cell. Methodsof conjugating therapeutic agents to antibodies is well-known in theart. Different antibodies have to be made for different types of cancer;for example, Rituximab recognizes CD20 protein on the outside of nonHodgkin's lymphoma cells; ADEPT is a treatment using antibodies thatrecognize bowel (colon) cancer; and Trastuzumab (Herceptin) recognizesbreast cancer cells that produce too much of the protein HER 2 (“HER 2positive”). Other antibodies include, for example, Tositumomab (Bexxar),Cetuximab (Erbitux), Ibritumomab (Zevalin), Alemtuzumab (Campath-1H),Epratuzumab (Lymphocide), Gemtuzumab ozogamicin (Mylotarg) and Bevacimab(Avastin). Thus, the viruses provided herein can be administeredconcurrently with, or sequentially to, one or more monoclonal antibodiesin the treatment of cancer. In one embodiment, additional therapy isadministered in the form of one or more of any of the other treatmentmodalities provided herein.

Rather than attempting to prevent infection, such as is the case withthe influenza virus, cancer vaccines help treat the cancer once it hasdeveloped. The aim of cancer vaccines is to stimulate the immuneresponse. Cancer vaccines include, for example, antigen vaccines, wholecell vaccines, dendritic cell vaccines, DNA vaccines and anti-idiotypevaccines. Antigen vaccines are vaccines made from tumor-associatedantigens in, or produced by, cancer cells. Antigen vaccines stimulate asubject's immune system to attack the cancer. Whole cell vaccines arevaccines that use the whole cancer cell, not just a specific antigenfrom it, to make the vaccine. The vaccine is made from a subject's owncancer cells, another subject's cancer cells or cancer cells grown in alaboratory. The cells are treated in the laboratory, usually withradiation, so that they can't grow, and are administered to the subjectvia injection or through an intravenous drip into the bloodstream sothey can stimulate the immune system to attack the cancer. One type ofwhole cell vaccine is a dendritic cell vaccine, which help the immunesystem to recognize and attack abnormal cells, such as cancer cells.Dendritic cell vaccines are made by growing dendritic cells alongsidethe cancer cells in the lab. The vaccine is administered to stimulatethe immune system to attack the cancer. Anti-idiotype vaccines arevaccines that stimulate the body to make antibodies against cancercells. Cancer cells make some tumor-associated antigens that the immunesystem recognizes as foreign. But because cancer cells are similar tonon-cancer cells, the immune system can respond weakly. DNA vaccinesboost the immune response. DNA vaccines are made from DNA from cancercells that carry the genes for the tumor-associated antigens. When a DNAvaccine is injected, it enables the cells of the immune system torecognize the tumor-associated antigens, and activates the cells in theimmune system (i.e., breaking tolerance). The most promising resultsfrom using DNA vaccines are in treating melanoma. Thus, the virusesprovided herein can be administered concurrently with, or sequentiallyto, a whole cell vaccine in the treatment of cancer. In one embodiment,additional therapy is administered in the form of one or more of any ofthe other treatment modalities provided herein.

Growth factors are natural substances that stimulate the bone marrow tomake blood cells. Recombinant technology can be used to generate growthfactors which can be administered to a subject to increase the number ofwhite blood cells, red blood cells and stem cells in the blood. Growthfactors used in cancer treatment to boost white blood cells includeGranulocyte Colony Stimulating Factor (G-CSF) also called filgrastim(Neupogen) or lenograstim (Granocyte) and Granulocyte and MacrophageColony Stimulating Factor (GM-CSF), also called molgramostim. A growthfactor to help treat anemia is erythropoietin (EPO). EPO encourages thebody to make more red blood cells, which in turn, increases hemoglobinlevels and the levels of oxygen in body tissues. Other growth factorsare being developed which can boost platelets. Thus, the virusesprovided herein can be administered concurrently with, or sequentiallyto, a growth factor such as GM-CSF, in the treatment of cancer. In oneembodiment, additional therapy is administered in the form of one ormore of any of the other treatment modalities provided herein.

Cancer growth inhibitors use cell-signaling molecules which control thegrowth and multiplication of cells, such as cancer cells. Drugs thatblock these signaling molecules can stop cancers from growing anddividing. Cancer growth factors include, but are not limited to,tyrosine kinases. Thus, drugs that block tyrosine kinases are tyrosinekinase inhibitors (TKIs). Examples of TKIs include, but are not limitedto, Erlotinib (Tarceva, OSI-774), Iressa (Gefitinib, ZD 1839) andImatinib (Glivec, STI 571). Another type of growth inhibitor isBortezomib (Velcade) for multiple myeloma and for some other cancers.Velcade is a proteasome inhibitor. Proteasomes are found in all cellsand help break down proteins in cells. Interfering with the action ofproteosomes causes a build up of proteins in the cell to toxic levels;thereby killing the cancer cells. Cancer cells are more sensitive toVelcade than normal cells. Thus, the viruses provided herein can beadministered concurrently with, or sequentially to, a cancer growthinhibitor, such as Velcade, in the treatment of cancer. In oneembodiment, additional therapy is administered in the form of one ormore of any of the other treatment modalities provided herein.

Cancers need a blood supply to expand and grow their own blood vesselsas they get bigger. Without its own blood supply, a cancer cannot growdue to lack of nutrients and oxygen. Anti-angiogenic drugs stop tumorsfrom developing their own blood vessels. Examples of these types ofdrugs include, but are not limited to, Thalidomide, mainly for treatingmyeloma but also in trials for other types of cancer, and Bevacizumab(Avastin), a type of monoclonal antibody that has been investigated forbowel cancer. Thus, the viruses provided herein can be administeredconcurrently with, or sequentially to, an anti-angiogenic drug in thetreatment of cancer. In one embodiment, additional therapy isadministered in the form of one or more of any of the other treatmentmodalities provided herein.

Interferon-alpha (IFN-α) is a natural substance produced in the body, invery small amounts, as part of the immune response. IFN-α isadministered as a treatment to boost the immune system and help fightcancers such as renal cell (kidney) cancer, malignant melanoma, multiplemyeloma and some types of leukemias. IFN-α works in several ways: it canhelp to stop cancer cells growing, it can also boost the immune systemto help it attack the cancer, and it can affect the blood supply to thecancer cells. Thus, the viruses provided herein can be administeredconcurrently with, or sequentially to, IFN-α in the treatment of cancer.In one embodiment, additional therapy is administered in the form of oneor more of any of the other treatment modalities provided herein.

Administration of IL-2 is a biological therapy drug because it isnaturally produced by the immune system. Thus, it is also animmunotherapy. Interleukin 2 is used in treating renal cell (kidney)cancer, and is being tested in clinical trials for several other typesof cancers. IL-2 works directly on cancer cells by interfering with cellgrow and proliferation; it stimulates the immune system by promoting thegrowth of killer T cells and other cells that attack cancer cells; andit also stimulates cancer cells to secrete chemoattractants that attractimmune system cells. IL-2 is generally administered as a subcutaneousinjection just under the skin once daily for 5 days, followed by 2 daysrest. The cycle of injections is repeated for 4 weeks followed by a weekwithout treatment. The treatment regiment and the number of cyclesadministered depends on the type of cancer and how it responds to thetreatment. IL-2 can be self-administered or administered by a healthprofessional. Alternatively, IL-2 can be administered intravenously viainjection or drip. Thus, the viruses provided herein can be administeredconcurrently with, or sequentially to, IL-2 in the treatment of cancer.In one embodiment, additional therapy is administered in the form of oneor more of any of the other treatment modalities provided herein.

Gene therapy involves treating cancer by blocking abnormal genes incancer cells, repairing or replacing abnormal genes in cancer cells,encouraging even more genes to become abnormal in cancer cells so thatthey die or become sensitive to treatment, using viruses to carrytreatment-activating enzymes into the cancer cells, or a combinationthereof. As a result, cancer cells die due to damage in the cell. Cancercells develop as a result of several types of mutations in several oftheir genes. Targeted genes include, but are not limited to, those thatencourage the cell to multiply (i.e., oncogenes), genes that stop thecell multiplying (i.e., tumor suppressor genes) and genes that repairother damaged genes. Gene therapy can involve repair of damagedoncogenes or blocking the proteins that the oncogenes produce. The tumorsuppressor gene, p53, is damaged in many human cancers. Viruses havebeen used in to deliver an undamaged p53 gene into cancer cells, andearly clinical trials are now in progress looking at treating cancerswith modified p53-producing viruses. Gene therapy could be used toreplace the damaged DNA repairing genes. In an alternative embodiment,methods of increasing DNA damage within a tumor cell can promote deathof the tumor cell or cause increased susceptibility of the tumor cell toother cancer treatments, such as radiotherapy or chemotherapy. Thus, theviruses provided herein can be administered concurrently with, orsequentially to, any of the gene therapy methods provided herein orknown in the art in the treatment of cancer. In one embodiment,additional therapy is administered in the form of one or more of any ofthe other treatment modalities provided herein.

Treatment of early stage bladder cancer is called intravesicaltreatment, which is mainly used to treat stage T1 bladder cancers thatare high grade (grade 3 or G3) or carcinoma in situ of the bladder (alsoknown as T is or CIS). BCG is a vaccine for tuberculosis (TB), whichalso has been found to be effective in treating CIS and preventingbladder cancers from recurring. In some cases, BCG vaccines have beenused for treating grade 2 early bladder cancer. Because bladder cancercan occur anywhere in the bladder lining, it cannot be removed in thesame way as the papillary early bladder cancers. Rather a BCG vaccine isadministered using intravesical therapy; that is, first, a catheter(tube) put is inserted into the bladder, followed by intra-catheteradministration of a BCG vaccine and/or a chemotherapy. BCG treatmentoccurs weekly for 6 weeks or more depending on the effect on the bladdercancer. BCG treatment of bladder cancer can be combined with other typesof treatments, such as administration of chemotherapy (intravesical),IL-2, treatment with drugs that make cells sensitive to light, vitamins,and photodynamic therapy. Thus, the viruses provided herein can beadministered concurrently with, or sequentially to, BCG vaccines in thetreatment of cancer. In one embodiment, additional therapy isadministered in the form of one or more of any of the other treatmentmodalities provided herein.

f. State of Subject

In another embodiment, the methods provided herein for administering avirus to a subject can be performed on a subject in any of a variety ofstates, including an anesthetized subject, an alert subject, a subjectwith elevated body temperature, a subject with reduced body temperature,or other state of the subject that is known to affect the accumulationof a virus in the tumor. As provided herein, it has been determined thata subject that is anesthetized can have a decreased rate of accumulationof a virus in a tumor relative to a subject that is not anesthetized.Further provided herein, it has been determined that a subject withdecreased body temperature can have a decreased rate of accumulation ofa virus in a tumor relative to a subject with a normal body temperature.Accordingly, provided herein are methods of administering a virus to asubject, where the methods can include administering a virus to asubject where the subject is not under anesthesia, such as generalanesthesia; for example, the subject can be under local anesthesia, orcan be unanesthetized. Also provided herein are methods of administeringa virus to a subject, where the methods can include administering avirus to a subject with altered body temperature, where the alterationof the body temperature can influence the ability of the virus toaccumulate in a tumor; typically, a decrease in body temperature candecrease the ability of a virus to accumulate in a tumor. Thus, in oneexemplary embodiment, a method is provided for administering a virus toa subject, where the method includes elevating the body temperature ofthe subject to a temperature above normal, and administering a virus tothe subject, where the virus can accumulate in the tumor more readily inthe subject with higher body temperature relative to the ability of thevirus to accumulate in a tumor of a subject with a normal bodytemperature. In another embodiment, localized elevations in temperaturein the area surrounding the tumor can be used to increase theaccumulation of the virus in the tumor.

2. Monitoring

The methods provided herein can further include one or more steps ofmonitoring the subject, monitoring the tumor, and/or monitoring thevirus administered to the subject. Any of a variety of monitoring stepscan be included in the methods provided herein, including, but notlimited to, monitoring tumor size, monitoring anti-(tumor antigen)antibody titer, monitoring the presence and/or size of metastases,monitoring the subject's lymph nodes, monitoring the subject's weight orother health indicators including blood or urine markers, monitoringanti-(viral antigen) antibody titer, monitoring viral expression of adetectable gene product, and directly monitoring viral titer in a tumor,tissue or organ of a subject.

The purpose of the monitoring can be simply for assessing the healthstate of the subject or the progress of therapeutic treatment of thesubject, or can be for determining whether or not further administrationof the same or a different virus is warranted, or for determining whenor whether or not to administer a compound to the subject where thecompound can act to increase the efficacy of the therapeutic method, orthe compound can act to decrease the pathogenicity of the virusadministered to the subject.

a. Monitoring Viral Gene Expression

In some embodiments, the methods provided herein can include monitoringone or more virally expressed genes. Viruses, such as those providedherein or otherwise known in the art, can express one or more detectablegene products, including but not limited to, detectable proteins.

As provided herein, measurement of a detectable gene product expressedby a virus can provide an accurate determination of the level of viruspresent in the subject. As further provided herein, measurement of thelocation of the detectable gene product, for example, by imaging methodsincluding, but not limited to, magnetic resonance, fluorescence, andtomographic methods, can determine the localization of the virus in thesubject. Accordingly, the methods provided herein that includemonitoring a detectable viral gene product can be used to determine thepresence or absence of the virus in one or more organs or tissues of asubject, and/or the presence or absence of the virus in a tumor ormetastases of a subject. Further, the methods provided herein thatinclude monitoring a detectable viral gene product can be used todetermine the titer of virus present in one or more organs, tissues,tumors or metastases. Methods that include monitoring the localizationand/or titer of viruses in a subject can be used for determining thepathogenicity of a virus; since viral infection, and particularly thelevel of infection, of normal tissues and organs can indicate thepathogenicity of the probe, methods of monitoring the localizationand/or amount of viruses in a subject can be used to determine thepathogenicity of a virus. Since methods provided herein can be used tomonitor the amount of viruses at any particular location in a subject,the methods that include monitoring the localization and/or titer ofviruses in a subject can be performed at multiple time points, and,accordingly can determine the rate of viral replication in a subject,including the rate of viral replication in one or more organs or tissuesof a subject; accordingly, the methods of monitoring a viral geneproduct can be used for determining the replication competence of avirus. The methods provided herein also can be used to quantitate theamount of virus present in a variety of organs or tissues, and tumors ormetastases, and can thereby indicate the degree of preferentialaccumulation of the virus in a subject; accordingly, the viral geneproduct monitoring methods provided herein can be used in methods ofdetermining the ability of a virus to accumulate in tumor or metastasesin preference to normal tissues or organs. Since the viruses used in themethods provided herein can accumulate in an entire tumor or canaccumulate at multiple sites in a tumor, and can also accumulate inmetastases, the methods provided herein for monitoring a viral geneproduct can be used to determine the size of a tumor or the number ofmetastases that are present in a subject. Monitoring such presence ofviral gene product in tumor or metastasis over a range of time can beused to assess changes in the tumor or metastasis, including growth orshrinking of a tumor, or development of new metastases or disappearanceof metastases, and also can be used to determine the rate of growth orshrinking of a tumor, or development of new metastases or disappearanceof metastases, or the change in the rate of growth or shrinking of atumor, or development of new metastases or disappearance of metastases.Accordingly, the methods of monitoring a viral gene product can be usedfor monitoring a neoplastic disease in a subject, or for determining theefficacy of treatment of a neoplastic disease, by determining rate ofgrowth or shrinking of a tumor, or development of new metastases ordisappearance of metastases, or the change in the rate of growth orshrinking of a tumor, or development of new metastases or disappearanceof metastases.

Any of a variety of detectable proteins can be detected in themonitoring methods provided herein; an exemplary, non-limiting list ofsuch detectable proteins includes any of a variety of fluorescentproteins (e.g., green or red fluorescent proteins), any of a variety ofluciferases, transferrin or other iron binding proteins; or receptors,binding proteins, and antibodies, where a compound that specificallybinds the receptor, binding protein or antibody can be a detectableagent or can be labeled with a detectable substance (e.g., aradionuclide or imaging agent). Viruses expressing a detectable proteincan be detected by a combination of the method provided herein and knowin the art. Viruses expressing more than one detectable protein or twoor more viruses expressing various detectable protein can be detectedand distinguished by dual imaging methods. For example, a virusexpressing a fluorescent protein and an iron binding protein can bedetected in vitro or in vivo by low light fluorescence imaging andmagnetic resonance, respectively. In another example, a virus expressingtwo or more fluorescent proteins can be detected by fluorescence imagingat different wavelength. In vivo dual imaging can be performed on asubject that has been administered a virus expressing two or moredetectable gene products or two or more viruses each expressing one ormore detectable gene products.

b. Monitoring Tumor Size

Also provided herein are methods of monitoring tumor and/or metastasissize and location. Tumor and or metastasis size can be monitored by anyof a variety of methods known in the art, including external assessmentmethods or tomographic or magnetic imaging methods. In addition to themethods known in the art, methods provided herein, for example,monitoring viral gene expression, can be used for monitoring tumorand/or metastasis size.

Monitoring size over several time points can provide informationregarding the increase or decrease in size of a tumor or metastasis, andcan also provide information regarding the presence of additional tumorsand/or metastases in the subject. Monitoring tumor size over severaltime points can provide information regarding the development of aneoplastic disease in a subject, including the efficacy of treatment ofa neoplastic disease in a subject.

c. Monitoring Antibody Titer

The methods provided herein also can include monitoring the antibodytiter in a subject, including antibodies produced in response toadministration of a virus to a subject. The viruses administered in themethods provided herein can elicit an immune response to endogenousviral antigens. The viruses administered in the methods provided hereinalso can elicit an immune response to exogenous genes expressed by avirus. The viruses administered in the methods provided herein also canelicit an immune response to tumor antigens. Monitoring antibody titeragainst viral antigens, viral expressed exogenous gene products, ortumor antigens can be used in methods of monitoring the toxicity of avirus, monitoring the efficacy of treatment methods, or monitoring thelevel of gene product or antibodies for production and/or harvesting.

In one embodiment, monitoring antibody titer can be used to monitor thetoxicity of a virus. Antibody titer against a virus can vary over thetime period after administration of the virus to the subject, where atsome particular time points, a low anti-(viral antigen) antibody titercan indicate a higher toxicity, while at other time points a highanti-(viral antigen) antibody titer can indicate a higher toxicity. Theviruses used in the methods provided herein can be immunogenic, and can,therefore, elicit an immune response soon after administering the virusto the subject. Generally, a virus against which a subject's immunesystem can quickly mount a strong immune response can be a virus thathas low toxicity when the subject's immune system can remove the virusfrom all normal organs or tissues. Thus, in some embodiments, a highantibody titer against viral antigens soon after administering the virusto a subject can indicate low toxicity of a virus. In contrast, a virusthat is not highly immunogenic can infect a host organism withouteliciting a strong immune response, which can result in a highertoxicity of the virus to the host. Accordingly, in some embodiments, ahigh antibody titer against viral antigens soon after administering thevirus to a subject can indicate low toxicity of a virus.

In other embodiments, monitoring antibody titer can be used to monitorthe efficacy of treatment methods. In the methods provided herein,antibody titer, such as anti-(tumor antigen) antibody titer, canindicate the efficacy of a therapeutic method such as a therapeuticmethod to treat neoplastic disease. Therapeutic methods provided hereincan include causing or enhancing an immune response against a tumorand/or metastasis. Thus, by monitoring the anti-(tumor antigen) antibodytiter, it is possible to monitor the efficacy of a therapeutic method incausing or enhancing an immune response against a tumor and/ormetastasis. The therapeutic methods provided herein also can includeadministering to a subject a virus that can accumulate in a tumor andcan cause or enhance an anti-tumor immune response. Accordingly, it ispossible to monitor the ability of a host to mount an immune responseagainst viruses accumulated in a tumor or metastasis, which can indicatethat a subject has also mounted an anti-tumor immune response, or canindicate that a subject is likely to mount an anti-tumor immuneresponse, or can indicate that a subject is capable of mounting ananti-tumor immune response.

In other embodiments, monitoring antibody titer can be used formonitoring the level of gene product or antibodies for production and/orharvesting. As provided herein, methods can be used for producingproteins, RNA molecules or other compounds by expressing an exogenousgene in a virus that has accumulated in a tumor. Further provided hereinare methods for producing antibodies against a protein, RNA molecule orother compound produced by exogenous gene expression of a virus that hasaccumulated in a tumor. Monitoring antibody titer against the protein,RNA molecule or other compound can indicate the level of production ofthe protein, RNA molecule or other compound by the tumor-accumulatedvirus, and also can directly indicate the level of antibodies specificfor such a protein, RNA molecule or other compound.

d. Monitoring General Health Diagnostics

The methods provided herein also can include methods of monitoring thehealth of a subject. Some of the methods provided herein are therapeuticmethods, including neoplastic disease therapeutic methods. Monitoringthe health of a subject can be used to determine the efficacy of thetherapeutic method, as is known in the art. The methods provided hereinalso can include a step of administering to a subject a virus.Monitoring the health of a subject can be used to determine thepathogenicity of a virus administered to a subject. Any of a variety ofhealth diagnostic methods for monitoring disease such as neoplasticdisease, infectious disease, or immune-related disease can be monitored,as is known in the art. For example, the weight, blood pressure, pulse,breathing, color, temperature or other observable state of a subject canindicate the health of a subject. In addition, the presence or absenceor level of one or more components in a sample from a subject canindicate the health of a subject. Typical samples can include blood andurine samples, where the presence or absence or level of one or morecomponents can be determined by performing, for example, a blood panelor a urine panel diagnostic test. Exemplary components indicative of asubject's health include, but are not limited to, white blood cellcount, hematocrit, or reactive protein concentration.

e. Monitoring Coordinated with Treatment

Also provided herein are methods of monitoring a therapy, wheretherapeutic decisions can be based on the results of the monitoring.Therapeutic methods provided herein can include administering to asubject a virus, where the virus can preferentially accumulate in atumor and/or metastasis, and where the virus can cause or enhance ananti-tumor immune response. Such therapeutic methods can include avariety of steps including multiple administrations of a particularvirus, administration of a second virus, or administration of atherapeutic compound. Determination of the amount, timing or type ofvirus or compound to administer to the subject can be based on one ormore results from monitoring the subject. For example, the antibodytiter in a subject can be used to determine whether or not it isdesirable to administer a virus or compound, the quantity of virus orcompound to administer, and the type of virus or compound to administer,where, for example, a low antibody titer can indicate the desirabilityof administering additional virus, a different virus, or a therapeuticcompound such as a compound that induces viral gene expression. Inanother example, the overall health state of a subject can be used todetermine whether or not it is desirable to administer a virus orcompound, the quantity of virus or compound to administer, and the typeof virus or compound to administer, where, for example, determining thatthe subject is healthy can indicate the desirability of administeringadditional virus, a different virus, or a therapeutic compound such as acompound that induces viral gene expression. In another example,monitoring a detectable virally expressed gene product can be used todetermine whether or not it is desirable to administer a virus orcompound, the quantity of virus or compound to administer, and the typeof virus or compound to administer. Such monitoring methods can be usedto determine whether or not the therapeutic method is effective, whetheror not the therapeutic method is pathogenic to the subject, whether ornot the virus has accumulated in a tumor or metastasis, and whether ornot the virus has accumulated in normal tissues or organs. Based on suchdeterminations, the desirability and form of further therapeutic methodscan be derived.

In one embodiment, determination of whether or not a therapeutic methodis effective can be used to derive further therapeutic methods. Any of avariety of methods of monitoring can be used to determine whether or nota therapeutic method is effective, as provided herein or otherwise knownin the art. If monitoring methods indicate that the therapeutic methodis effective, a decision can be made to maintain the current course oftherapy, which can include further administrations of a virus orcompound, or a decision can be made that no further administrations arerequired. If monitoring methods indicate that the therapeutic method isineffective, the monitoring results can indicate whether or not a courseof treatment should be discontinued (e.g., when a virus is pathogenic tothe subject), or changed (e.g., when a virus accumulates in a tumorwithout harming the host organism, but without eliciting an anti-tumorimmune response), or increased in frequency or amount (e.g., when littleor no virus accumulates in tumor).

In one example, monitoring can indicate that a virus is pathogenic to asubject. In such instances, a decision can be made to terminateadministration of the virus to the subject, to administer lower levelsof the virus to the subject, to administer a different virus to asubject, or to administer to a subject a compound that reduces thepathogenicity of the virus. In one example, administration of a virusthat is determined to be pathogenic can be terminated. In anotherexample, the dosage amount of a virus that is determined to bepathogenic can be decreased for subsequent administration; in oneversion of such an example, the subject can be pre-treated with anothervirus that can increase the ability of the pathogenic virus toaccumulate in tumor, prior to re-administering the pathogenic virus tothe subject. In another example, a subject can have administered theretoa virus that is pathogenic to the subject; administration of such apathogenic virus can be accompanied by administration of, for example,an antiviral compound (e.g., cidofovir), pathogenicity attenuatingcompound (e.g., a compound that down-regulates the expression of a lyticor apoptotic gene product), or other compound that can decrease theproliferation, toxicity, or cell killing properties of a virus, asdescribed herein elsewhere. In one variation of such an example, thelocalization of the virus can be monitored, and, upon determination thatthe virus is accumulated in tumor and/or metastases but not in normaltissues or organs, administration of the antiviral compound orpathogenicity attenuating compound can be terminated, and the pathogenicactivity of the virus can be activated or increased, but limited to thetumor and/or metastasis. In another variation of such an example, afterterminating administration of the antiviral compound or pathogenicityattenuating compound, the presence of the virus and/or pathogenicity ofthe virus can be further monitored, and administration of such acompound can be reinitiated if the virus is determined to pose a threatto the host by, for example, spreading to normal organs or tissues,releasing a toxin into the vasculature, or otherwise having pathogeniceffects reaching beyond the tumor or metastasis.

In another example, monitoring can determine whether or not a virus hasaccumulated in a tumor or metastasis of a subject. Upon such adetermination, a decision can be made to further administer additionalvirus, a different virus or a compound to the subject. In anotherexample, monitoring the presence of a virus in a tumor can be used indeciding to administer to the subject a compound, where the compound canincrease the pathogenicity, proliferation, or immunogenicity of a virusor the compound can otherwise act in conjunction with the virus toincrease the proliferation, toxicity, tumor cell killing, or immuneresponse eliciting properties of a virus; in one variation of such anexample, the virus can, for example, have little or no lytic or cellkilling capability in the absence of such a compound; in a furthervariation of such an example, monitoring of the presence of the virus ina tumor or metastasis can be coupled with monitoring the absence of thevirus in normal tissues or organs, where the compound is administered ifthe virus is present in tumor or metastasis and not at all present orsubstantially not present in normal organs or tissues; in a furthervariation of such an example, the amount of virus in a tumor ormetastasis can be monitored, where the compound is administered if thevirus is present in tumor or metastasis at sufficient levels.

J. METHODS OF PRODUCING GENE PRODUCTS AND ANTIBODIES

Provided herein are viruses, and methods for making and using suchviruses for production products of exogenous genes and/or for productionof antibodies specific for exogenous gene products. The methods providedherein result in efficient recombinant production of biologically activeproteins. As provided herein, a system based on the accumulation ofviruses in tumors can be used for simple, quick, and inexpensiveproduction of proteins and other biological compounds originating fromcloned nucleotide sequences. This system also is useful for theconcomitant overproduction of polypeptides, RNA or other biologicalcompounds (in tumor tissue) and antibodies against those compounds (inthe serum) in the same animal. These systems have the followingadvantages: (a) the viruses target the tumor specifically withoutaffecting normal tissue; (b) the expression and secretion of thetherapeutic gene constructs can be, optionally, under the control of aninducible promoter enabling secretion to be switched on or off; and (c)the location of the delivery system inside the tumor can be verified bydirect visualization before activating gene expression and proteindelivery.

As provided herein, after administration, a virus such as vaccinia viruscan enter the tumor of an animal and, due to the immunoprivileged stateof the tumor, can replicate preferentially in the tumor tissues andthereby can overproduce the inserted gene encoded protein in the tumors.After harvesting the tumor tissues, the localized and over-expressedprotein can be isolated by a simple procedure from tumor homogenates. Inaddition, based on findings that only 0.2 to 0.3% of the desiredproteins produced in the tumor are found in the blood stream of the sameanimal, a simultaneous vaccination of the mouse and efficient antibodyproduction against the overproduced protein can be achieved. Thus, serumfrom the same mouse (or any other animal) can be harvested and used asmouse-derived antibodies against the proteins or other productsoverproduced in the tumor.

Thus, provided herein are methods of producing gene products and/orantibodies in a non-human subject, by administering to a subjectcontaining a tumor, a virus, wherein the virus expresses a gene encodinga selected protein or RNA to be produced, a protein or RNA whoseexpression can result in the formation of a compound to be produced, ora selected protein or RNA against which an antibody is to be produced.The gene or genes expressed can be endogenous or exogenous to the virus.The nucleotide sequences can be contained in a recombinant viruscontaining appropriate expression cassettes. For example, the nucleotidesequences can be operatively linked with a promoter allowing highexpression. Such promoters can include, for example, induciblepromoters; a variety of such promoters are known to persons skilled inthe art. Expression of the gene(s) can be regulated, for example, by atranscriptional activator or inducer, or a transcriptional suppressor.In one embodiment, the methods provided herein for producing a protein,RNA, compound or antibody can further include monitoring thelocalization and/or level of the virus in the subject by detecting adetectable protein, wherein the detectable protein can indicate theexpression of the selected gene, or can indicate the readiness of thevirus to be induced to express the selected gene or for suppression ofexpression of the gene to be terminated or suspended. In one embodiment,the virus contains a nucleotide sequence encoding a detectable protein,such as a luminescent or fluorescent protein, or a protein capable ofinducing a detectable signal.

The virus can be administered to a transgenic animal or a non-transgenicanimal. The subject can be selected according to its ability topost-translationally process the selected protein.

In one embodiment, methods are provided for producing a desiredpolypeptide, RNA or compound, the method including the following steps:(a) injecting a virus containing a nucleotide sequence encoding thedesired polypeptide or RNA into an animal bearing a tumor; (b)harvesting the tumor tissue from the animal; and (c) isolating thedesired polypeptide, RNA or compound from the tumor tissue.

Steps of an exemplary method can be summarized as follows (shown for aparticular embodiment, for example a vaccinia virus, additionallycontaining a gene encoding a light-emitting protein):

(1) Insertion of the desired DNA or cDNA into the vaccinia virus genome;(2) modification of the vaccinia virus genome with light-emittingprotein construct as expression marker;(3) recombination and virus assembly in cell culture;(4) screening of individual viral particles carrying inserts followed bylarge scale virus production and concentration;(5) administration of the viral particles into mice or other animalsbearing tumors of human, non-human primate or other mammalian origins;(6) verification of viral replication and protein overproduction inanimals based on light emission;(7) harvest of tumor tissues and, optionally, the blood (separately);and(8) purification of over-expressed proteins from tumors and, optionally,antisera from blood using conventional methods.

Any viruses can be used in the methods provided herein, provided thatthey replicate in the animal, are not pathogenic for the animal, forexample, are attenuated, and/or are recognized by the immune system ofthe animal. In some embodiments, such viruses also can express exogenousgenes. Suitable viruses and cells are, for example, disclosed in EP A1 1281 772 and EP A1 1 281 767. The person skilled in the art also knowshow to generate animals carrying the desired tumor (see, for example, EPA1 1 281 767 or EP A1 1 281 777).

Also provided is a method for simultaneously producing a desiredpolypeptide, RNA or compound and an antibody directed to thepolypeptide, RNA or compound, the method having the following steps: (a)administering a virus containing a nucleotide sequence encoding thedesired polypeptide or RNA into an animal bearing a tumor; (b)harvesting the tumor tissue from the animal; (c) isolating the desiredpolypeptide, RNA or compound from the tumor tissue; and (d) isolatingthe antibody directed to the polypeptide, RNA or compound from the serumobtained from the animal. This approach can be used for generatingpolypeptides and/or antibodies against the polypeptides which are toxicor unstable, or which require species specific cellular environment forcorrect folding or modifications.

A person skilled in the art is familiar with a variety of viralexpression vectors, which can be selected according to the virus used toinfect the tumor, the cell type of the tumor, the organism to beinfected, and other factors known in the art. Suitable viruses for useherein, include, but are not limited to, poxvirus, adenovirus, herpessimplex virus, Newcastle disease virus, vesicular stomatitis virus,mumps virus, influenza virus, measles virus, reovirus, humanimmunodeficiency virus, hanta virus, myoma virus, cytomegalovirus, andlentivirus. In some embodiments, virus can be a vaccinia virus,including the vaccinia viruses disclosed herein.

For generating protein or RNA-encoding nucleotide sequences and forconstructing expression vectors or viruses that contain the nucleotidesequences, it is possible to use general methods known in the art. Thesemethods include, for example, in vitro recombination techniques,synthetic methods and in vivo recombination methods as known in the art,and exemplified in Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd edition (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

In some embodiments, the protein or RNA to be produced in the tumor canbe linked to an inducible promoter, such as a promoter that can beinduced by a substance endogenous to the subject, or by a substance thatcan be administered to a subject. Accordingly, provided herein aremethods of producing a protein or RNA in a tumor, where the productioncan be induced by administration of a substance to a subject, and,optionally, harvesting the tumor and isolating the protein or RNA fromthe tumor. Such induction methods can be coupled with methods ofmonitoring a virus in a subject. For example, a virus can be monitoredby detecting a detectable protein. In methods that include monitoring,detection of a desired localization and/or level of virus in the subjectcan be coordinated with induction of viral gene expression. For example,when a virally expressed detectable protein is detected in tumor, butnot appreciably in normal organs or tissues, an inducer can beadministered to the subject. In another example, when a virallyexpressed detectable protein is detected in tumor, and also in normalorgans or tissues, administration of an inducer can be suspended orpostponed until the detectable protein is no longer detected in normalorgans or tissues. In another example, when a virally expresseddetectable protein is detected at sufficient levels in tumor, an inducercan be administered to the subject. In another example, when a virallyexpressed detectable protein is not detected at sufficient levels intumor administration of an inducer can be suspended or postponed untilthe detectable protein is detected at sufficient levels in the tumor.

Also provided herein are methods of producing a protein or RNA in atumor, by administering a virus encoding the protein or RNA, and asuppressor of gene expression. The suppressor of gene expression can beadministered for a pre-defined period of time, or until the virusaccumulates in tumor but not in normal organs or tissues, or untilsufficient levels of the virus have accumulated in the tumor, at whichpoint administration of the suppressor can be terminated or suspended,which can result in expression of the protein or RNA. As will berecognized by one skilled in the art, methods similar to those providedherein in regard to monitoring a detectable protein and administering aninducer, can also apply for terminating or suspending administration ofa suppressor.

Any of a variety of animals, including laboratory or livestock animalscan be used, including for example, mice, rats and other rodents,rabbits, guinea pigs, pigs, sheep, goats, cows and horses. Exemplaryanimals are mice. The tumor can be generated by implanting tumor cellsinto the animal. Generally, for the production of a desired polypeptide,RNA, or compound, any solid tumor type can be used, such as a fastgrowing tumor type. Exemplary fast growing tumor types include C6 ratglioma and HCT116 human colon carcinoma. Generally, for the productionof a desired antibody, a relatively slow growing tumor type can be used.Exemplary slow growing tumor types include HT1080 human fibrosarcoma andGI-101A human breast carcinoma. For T-independent antibody production,nu⁻/nu⁻ mice bearing allogenic tumor or xenografts can be used; whilefor T-dependent antibody production, immunocompetent mice with syngenictumors can be used. In some embodiments, such as where the compound tobe produced is a protein, the virus selected can be a virus that usesthe translational components (e.g., proteins, vesicles, substrates) ofthe tumor cells, such as, for example, a virus that uses thetranslational components of a tumor cell. In such instances, the tumorcell type can be selected according to the desired post-translationalprocessing to be performed on the protein, including proteolysis,glycosylation, lipidylation, disulfide formation, and any refolding ormultimer assembly that can require cellular components for completing.In some examples, the tumor cell type selected can be the same speciesas the protein to be expressed, thus resulting in species-specificpost-translational processing of the protein; an exemplary tumor celltype-expressed protein species is human.

1. Production of Recombinant Proteins and RNA Molecules

The tumor tissue can be surgically removed from the animal. Afterhomogenization of the tumor tissue, the desired polypeptide, RNA orother biological compound can be purified according to establishedmethods. For example, in the case of a recombinant polypeptide, thepolypeptide might contain a bindable tag such as a his-tag, and can bepurified, for example, via column chromatography. The time necessary foraccumulation of sufficient amounts of the polypeptide or RNA in thetumor of the animal depends on many factors, for example, the kind ofanimal or the kind of tumor, and can be determined by the skilled personby routine experimentation. In general, expression of the desiredpolypeptide can be detected two days after virus injection. Theexpression peaks approximately two weeks after injection, and lasts upto two months. In some embodiments, the amount of desired polypeptide orRNA in the tumor can be determined by monitoring a virally expresseddetectable substance, where the concentration of the detectablesubstance can reflect the amount of desired polypeptide or RNA in thetumor.

In another embodiment, the desired polypeptide, RNA or other compoundcan be manufactured in the subject, and provide a beneficial effect tothe subject. In one example, a virus can encode a protein or RNA, or aprotein that manufactures a compound that is not manufactured by thesubject. In one example, a virus can encode a peptide hormone orcytokine, such as insulin, which can be released into the vasculature ofa subject lacking the ability to produce insulin or requiring increasedinsulin concentrations in the vasculature. In another example, bloodclotting factors can be manufactured in a subject with blood clottingdeficiency, such as a hemophiliac. In some embodiments, the protein orRNA to be produced in the tumor can be linked to an inducible promoter,such as a promoter that can be induced by increased glucoseconcentrations. In such instances, the manufacture of the protein or RNAcan be controlled in response to one or more substances in the subjector by one or more substances that can be administered to a subject, suchas a compound that can induce transcription, for example, RU486. Thus,in some embodiments, the methods provided herein can includeadministering to a subject having a tumor, a virus that can express oneor more genes encoding a beneficial gene product or a gene product thatcan manufacture a beneficial compound.

2. Production of Antibodies

Also provided are methods for producing a desired antibody, the methodcomprising the following steps: (a) administering a virus containing anucleotide sequence encoding an antigen into an animal bearing a tumor;and (b) isolating the antibody directed to the antigen from the serumobtained from the animal. The antibodies directed to the antigen can beisolated and purified according to well known methods. Antibodies thatare directed against specific contaminating antigens can be removed byadsorption, and the antibodies directed against the target antigen canbe separated from contaminating antibodies by affinity purification, forexample, by immunoaffinity chromatography using the recombinant antigenas the ligand of the column, by methods known in the art. Antibodies canbe collected from the animal in a single harvest, or can be collectedover time by collection bleeds, as is known in the art.

K. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Generation of Modified Vaccinia Virus Strains A. Constructionof Modified Vaccinia Viruses

Modified vaccinia viruses were generated by replacing nucleic acid orinserting nucleic acid at several loci in the vaccinia virus genome asfollows: the F14.5L (also referred to as F3; see U.S. Patent PublicationNo. 2005/0031643), thymidine kinase (TK), hemagglutinin (HA) and A34Rgene loci (the A34R gene encodes a C-type lectin-like glycoprotein,gp22-24, that is present in the outer membrane of extracellularenveloped virus (EEV), and that is reported to be required forinfectivity of EEV; see, e.g., McIntosh et al. (1996) J. Virol.70:272081). The heterologous DNA inserted either was (1) a relativelyshort non-coding DNA fragment, (2) an expression cassette containingprotein-encoding DNA operably linked in the correct or reverseorientation to a vaccinia virus promoter, or (3) the coding sequence ofthe A34R gene (SEQ ID NO: 58) from vaccinia virus strain IHD-J.

The starting strain for the modified vaccinia viruses described hereinwas vaccinia virus (VV) strain GLV-1h68 (also named RVGL21, SEQ ID NO:1). This genetically engineered strain, which has been described in U.S.Patent Publication No. 2005/0031643, contains DNA insertions in theF14.5L, thymidine kinase (TK) and hemagglutinin (HA) genes. GLV-1h68 wasprepared from the vaccinia virus strain designated LIVP (a vacciniavirus strain, originally derived by adapting the Lister strain (ATCCCatalog No. VR-1549) to calf skin (Research Institute of ViralPreparations, Moscow, Russia, Al'tshtein et al. (1983) Dokl. Akad. NaukUSSR 285:696-699). The LIVP strain (whose genome sequence is set forthin SEQ ID NO: 2), from which GLV-1h68 was generated, contains a mutationin the coding sequence of the TK gene (see SEQ ID NO: 2 for the sequenceof the LIVP. strain) in which a substitution of a guanine nucleotidewith a thymidine nucleotide (nucleotide position 80207 of SEQ ID NO: 2)introduces a premature STOP codon within the coding sequence.

As described in U.S. Patent Publication No. 2005/0031643 (seeparticularly Example 1 of the application), GLV-1h68 was generated byinserting expression cassettes encoding detectable marker proteins intothe F14.5L (also designated in LIVP as F3) gene, thymidine kinase (TK)gene, and hemagglutinin (HA) gene loci of the vaccinia virus LIVPstrain. Specifically, an expression cassette containing a Ruc-GFP cDNA(a fusion of DNA encoding Renilla luciferase and DNA encoding GFP) underthe control of a vaccinia synthetic early/late promoter P_(SEL) wasinserted into the F14.5L gene; an expression cassette containing DNAencoding beta-galactosidase under the control of the vaccinia early/latepromoter P7.5 k (denoted (P_(7.5k))LacZ) and DNA encoding a rattransferrin receptor positioned in the reverse orientation fortranscription relative to the vaccinia synthetic early/late promoterP_(SEL) (denoted (P_(SEL))rTrfR) was inserted into the TK gene (theresulting virus does not express transferrin receptor protein since theDNA encoding the protein is positioned in the reverse orientation fortranscription relative to the promoter in the cassette); and anexpression cassette containing DNA encoding β-glucuronidase under thecontrol of the vaccinia late promoter P_(11k) (denoted (P_(11k))gusA)was inserted into the HA gene. Another genetically engineered vacciniastrain, designated GLV-1h22 was produced that has essentially the samegenotype as GLV-1h68, with the exception that, in the expressioncassette inserted into the TK gene (SEQ ID NO: 3), the DNA encoding therat transferrin receptor is in the correct orientation for transcriptionfrom the vaccinia synthetic early/late promoter P_(SEL). GLV-1h22 wasconstructed using the same method as used to create GLV-1h68, which isdescribed in detail in U.S. Patent Publication No. 2005/0031643, withexception that the expression cassette inserted into the TK locus wasgenerated using the pSC65-TfR transfer vector (also described in U.S.Patent Publication No. 2005/0031643; the parent vector for GLV-1h22 isRVGL19, which is shown in FIG. 1B and described in Example 1 of U.S.Patent Publication No. 2005/0031643).

Insertion of the expression cassettes into the LIVP genome in thegeneration of strains GLV-1h68 and GLV-1h22 resulted in disruption ofthe coding sequences for each of the F14.5L, TK and HA genes;accordingly, all three genes in the resulting strains are nonfunctionalin that they do not encode the corresponding full-length proteins. Asdescribed in U.S. Patent Publication No. 2005/0031643, disruption ofthese genes not only attenuates the virus but also enhances itstumor-specific accumulation. Previous data have shown that systemicdelivery of the GLV-1h68 virus in a mouse model of breast cancerresulted in the complete eradication of large subcutaneous GI-101A humanbreast carcinoma xenograft tumors in nude mice (see U.S. PatentPublication No. 2005/0031643).

1. Modified Viral Strains

Modified recombinant vaccinia viruses containing heterologous DNAinserted into one or more loci of the vaccinia virus genome weregenerated via homologous recombination between DNA sequences in thegenome and a transfer vector using methods described herein and known tothose of skill in the art (see, e.g., Falkner and Moss (1990) J. Virol.64:3108-2111; Chakrabarti et al. (1985) Mol. Cell. Biol. 5:3403-3409;and U.S. Pat. No. 4,722,848). In these methods, the existing target genein the starting vaccinia virus genome is replaced by an interrupted copyof the gene contained in the transfer vector through two crossoverevents: a first crossover event of homologous recombination between thevaccinia virus genome and the transfer vector and a second crossoverevent of homologous recombination between direct repeats within thetarget locus. The interrupted version of the target gene that is in thetransfer vector contains the insertion DNA flanked on each side by DNAcorresponding to the left portion of the target gene and right portionof the target gene, respectively. The transfer vector also contains adominant selection marker, e.g., the E. coli guaninephosphoribosyltransferase (gpt) gene, under the control of a vacciniavirus early promoter (e.g., P_(7.5kE)). Including such a marker in thevector enables a transient dominant selection process to identifyrecombinant virus grown under selective pressure that has incorporatedthe transfer vector within its genome. Because the marker gene is notstably integrated into the genome, it is deleted from the genome in asecond crossover event that occurs when selection is removed. Thus, thefinal recombinant virus contains the interrupted version of the targetgene as a disruption of the target loci, but does not retain theselectable marker from the transfer vector.

Homologous recombination between a transfer vector and a startingvaccinia virus genome occurred upon introduction of the transfer vectorinto cells that have been infected with the starting vaccinia virus. Aseries of transfer vectors was constructed as described below and thefollowing modified vaccinia strains were constructed: GLV-1i69,GLV-1h70, GLV-1h71, GLV-1h72, GLV-1h73, GLV-1h74, GLV-1h81, GLV-1h82,GLV-1h83, GLV-1h84, GLV-1h85, GLV-1h86, GLV-1j87, GLV-1j88, GLV-1j89,GLV-1h90, GLV-1h91, GLV-1h92, GLV-1h96, GLV-1h97, GLV-1h98, GLV-1h104,GLV-1h105, GLV-1h106, GLV-1h107, GLV-1h108 and GLV-1h109. Theconstruction of these strains is summarized in the following Table,which lists the modified vaccinia virus strains, including thepreviously described GLV-1h68, their respective genotypes, and thetransfer vectors used to engineer the viruses:

TABLE 2 Generation of engineered vaccinia viruses Name of Virus ParentalVirus VV Transfer Vector Genotype GLV-1h68 — — F14.5L: (P_(SEL))Ruc-GFPTK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA: (P_(11k))gusA GLV-1i69 GLV-1h68A34R gene from F14.5L: (P_(SEL))Ruc-GFP VV IHD-J TK:(P_(SEL))rTrfR-(P_(7.5k))LacZ HA: (P_(11k))gusA A34R: A34R-IHD-JGLV-1h70 GLV-1h68 pNCVVhaT F14.5L: (P_(SEL))Ruc-GFP TK:(P_(SEL))rTrfR-(P_(7.5k))LacZ HA: HindIII-BamHI GLV-1h71 GLV-1h68pNCVVf14.5lT F14.5L: BamHI-HindIII TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA:(P_(11k))gusA GLV-1h72 GLV-1h68 pCR-TKLR-gpt2 F14.5L: (P_(SEL))Ruc-GFPTK: SacI-BamHI HA: (P_(11k))gusA GLV-1h73 GLV-1h70 pNCVVf14.5lT F14.5L:BamHI-HindIII TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA: HindIII-BamHIGLV-1h74 GLV-1h73 pCR-TKLR-gpt2 F14.5L: BamHI-Hind III TK: SacI-BamHIHA: HindIII-BamHI GLV-1h81 GLV-1h68 pNCVVhaT-SEL-hk5 F14.5L:(P_(SEL))Ruc-GFP TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA: (P_(SEL))hk-5GLV-1h82 GLV-1h22 pNCVVhaT-ftn F14.5L: (P_(SEL))Ruc-GFP TK:(P_(SEL))TrfR-(P_(7.5k))LacZ HA: (P_(SEL))ftn GLV-1h83 GLV-1h68pNCVVhaT-ftn F14.5L: (P_(SEL))Ruc-GFP TK: (P_(SEL))rTrfR-(P_(7.5k))LacZHA: (P_(SEL))ftn GLV-1h84 GLV-1h73 pCR-TK-SEL-mRFP1 F14.5L: BamHI-HindIII TK: (P_(SEL))CBG99-mRFP1 HA: Hind III-BamHI GLV-1h85 GLV-1h72pNCVVf14.5lT F14.5L: BamHI-HindIII TK: Sac I-BamHI HA: (P_(11k))gusAGLV-1h86 GLV-1h72 pNCVVhaT F14.5L: (P_(SEL))Ruc-GFP TK: Sac I-BamHI HA:Hind III-BamHI GLV-1j87 GLV-1h68 pCR-gpt-dA35R6 F14.5L: (P_(SEL))Ruc-GFPTK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA: (P_(11k))gusA A35R: Multiplecloning sites (MCS) GLV-1j88 GLV-1h73 pCR-gpt-dA35R6 F14.5L:BamHI-HindIII TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA: HindIII-BamHI A35R:MCS GLV-1j89 GLV-1h74 pCR-gpt-dA35R6 F14.5L: BamHI-HindIII TK:SacI-BamHI HA: HindIII-BamHI A35R: MCS GLV-1h90 GLV-1h68 HA-SE-IL-6-1F14.5L: (P_(SEL))Ruc-GFP TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA:(P_(SE))sIL-6R/IL-6 GLV-1h91 GLV-1h68 HA-SEL-IL-6-1 F14.5L:(P_(SEL))Ruc-GFP TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA:(P_(SEL))sIL-6R/IL-6 GLV-1h92 GLV-1h68 HA-SL-IL-6-1 F14.5L:(P_(SEL))Ruc-GFP TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA:(P_(SL))sIL-6R/IL-6 GLV-1h96 GLV-1h68 FSE-IL-24 F14.5L: (P_(SE))IL-24TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA: (P_(11k))gusA GLV-1h97 GLV-1h68FSEL-IL-24 F14.5L: (P_(SEL))IL-24 TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA:(P_(11k))gusA GLV-1h98 GLV-1h68 FSL-IL-24 F14.5L: (P_(SL))IL-24 TK:(P_(SEL))rTrfR-(P_(7.5k))LacZ HA: (P_(11k))gusA GLV-1h104 GLV-1h68pCR-TK-SE-tTF- F14.5L: (P_(SEL))Ruc-GFP RGD TK: (P_(SE))tTF-RGD HA:(P_(11k))gusA GLV-1h105 GLV-1h68 pCR-TK-SEL-tTF- F14.5L:(P_(SEL))Ruc-GFP RGD TK: (P_(SEL))tTF-RGD HA: (P_(11k))gusA GLV-1h106GLV-1h68 pCR-TK-SL-tTF- F14.5L: (P_(SEL))Ruc-GFP RGD TK: (P_(SL))tTF-RGDHA: (P_(11k))gusA GLV-1h107 GLV-1h68 pCR-TK-SE-G6- F14.5L:(P_(SEL))Ruc-GFP FLAG TK: (P_(SE))G6-FLAG HA: (P_(11k))gusA GLV-1h108GLV-1h68 pCR-TK-SEL-G6- F14.5L: (P_(SEL))Ruc-GFP FLAG TK:(P_(SEL))G6-FLAG HA: (P_(11k))gusA GLV-1h109 GLV-1h68 pCR-TK-SL-G6-F14.5L: (P_(SEL))Ruc-GFP FLAG TK: (P_(SL))G6-FLAG HA: (P_(11k))gusABriefly, these strains were generated as follows (further details areprovided below):

GLV-1i69 was generated by replacement of the coding sequence of the A34Rgene in starting strain GLV-1h68 (nucleotides 153693 to 154199 in SEQ IDNO: 1) with the A34R gene from well-known vaccinia virus IHD-J strain.

GLV-1h70 was generated by insertion of a short non-coding DNA fragmentcontaining HindIII and BamHI sites into the HA locus of starting strainGLV-1h68 thereby deleting the gusA expression cassette at the HA locusof GLV-1h68. Thus, in strain GLV-1h70, the vaccinia HA gene isinterrupted within the coding sequence by a short non-coding DNAfragment.

GLV-1h71 was generated by insertion of a short non-coding DNA fragmentcontaining BamHI and HindIII sites (SEQ ID NO: 12) into the F14.5L locusof starting strain GLV-1h68 thereby deleting the Ruc-GFP fusion geneexpression cassette at the F14.5L locus of GLV-1h68. Thus, in strainGLV-1h71, the vaccinia F14.5L gene is interrupted within the codingsequence by a short non-coding DNA fragment.

GLV-1h72 was generated by insertion of a short non-coding DNA fragmentcontaining SacI and BamHI sites (SEQ ID NO: 18) into the TK locus ofstarting strain GLV-1h68 thereby deleting the LacZ/rTFr expressioncassette at the TK locus in GLV-1h68. Thus, in strain GLV-1h72, thevaccinia TK gene is interrupted within the coding sequence by a shortnon-coding DNA fragment.

GLV-1h73 was generated by insertion of a short non-coding DNA fragmentcontaining BamHI and HindIII sites (SEQ ID NO: 12) into the F14.5L locusof GLV-1h70 thereby deleting the Ruc-GFP fusion gene expression cassetteat the F14.5L locus of GLV-1h70. Thus, in strain GLV-1h73, the vacciniaHA and F14.5L genes are interrupted within the coding sequence by ashort non-coding DNA fragment.

GLV-1h74 was generated by insertion of a short non-coding DNA fragmentcontaining SacI and BamHI sites (SEQ ID NO: 18) into the TK locus ofstrain GLV-1h73 thereby deleting the LacZ/rTFr expression cassette atthe TK locus of GLV-1 h73. Thus, in strain GLV-1h74, the vaccinia HA,F14.5L and TK genes are interrupted within the coding sequence by ashort non-coding DNA fragment.

GLV-1h81 was generated by insertion of an expression cassette encodingthe plasminogen K5 domain under the control of the vaccinia P_(SEL)promoter into the HA locus of starting strain GLV-1h68 thereby deletingthe gusA expression cassette at the HA locus of starting GLV-1h68. Thus,in strain GLV-1h81, the vaccinia HA gene is interrupted within thecoding sequence by a DNA fragment containing DNA encoding theplasminogen K5 domain operably linked to the vaccinia syntheticearly/late promoter.

GLV-1h82 was generated by insertion of an expression cassette encodingE. coli ferritin under the control of the vaccinia P_(SEL) promoter intothe HA locus of strain GLV-1h22 thereby deleting the gusA expressioncassette at the HA locus of GLV-1h22. Thus, in strain GLV-1h82, thevaccinia HA gene is interrupted within the coding sequence by a DNAfragment containing DNA encoding E. coli ferritin operably linked to thevaccinia synthetic early/late promoter

GLV-1h83 was generated by insertion of an expression cassette encodingE. coli ferritin under the control of the vaccinia P_(SEL) promoter intothe HA locus of starting strain GLV-1h68 thereby deleting the gusAexpression cassette at the HA locus of GLV-1h68. Thus, in strainGLV-1h83, the vaccinia HA gene is interrupted within the coding sequenceby a DNA fragment containing DNA encoding E. coli ferritin operablylinked to the vaccinia synthetic early/late promoter.

GLV-1h84 was generated by insertion of an expression cassette containingDNA encoding CBG99 and mRFP1 connected through a picornavirus 2A elementand under the control of the vaccinia synthetic early/late promoter(P_(SEL)) into the TK locus of strain GLV-1h73 thereby deleting theLacZ/rTFr expression cassette at the TK locus of GLV-1h73. Thus, instrain GLV-1h84, the vaccinia HA and F14.5L genes are interrupted withinthe coding sequence by a short non-coding DNA fragment, and the vacciniaTK gene is interrupted within the coding sequence by DNA encoding afusion of CBG99 and mRFP1 proteins. Since DNAs encoding both markerproteins (CBG99 and mRFP1) are under the control of the same promoter,only one transcript is produced. During translation, these two proteinsare cleaved into two individual proteins at picornavirus 2A element(Osborn et al., Mol. Ther. 12: 569-74, 2005). CBG99 produces a morestable luminescent signal than does Renilla luciferase with a half-lifeof greater than 30 minutes, which makes both in vitro and in vivo assaysmore convenient. mRFP1 provides improvements in in vivo imaging relativeto GFP since mRFP1 can penetrate tissue deeper than GFP.

GLV-1h85 was generated by insertion of a short non-coding DNA fragmentcontaining BamHI and HindIII sites into the F14.5L locus of strainGLV-1h72 thereby deleting the Ruc-GFP fusion gene expression cassette atthe F14.5L locus of GLV-1h72. Thus, in strain GLV-1h85, the vacciniaF14.5L and TK genes are interrupted within the coding sequence by ashort non-coding DNA fragment.

GLV-1h86 was generated by insertion of a short non-coding DNA fragmentcontaining HindIII and BamHI sites into the HA locus of strain GLV-1h72thereby deleting the gusA expression cassette at the HA locus ofGLV-1h72. Thus, in strain GLV-1h86, the vaccinia TK and HA genes areinterrupted within the coding sequence by a short non-coding DNAfragment

GLV-1j87 was generated by deletion the coding sequence of the A35R genein starting strain GLV-1h68 (nucleotides 154,243 to 154,773 in SEQ IDNO: 1). Thus, in strain GLV-1j87, the vaccinia A35 gene is replaced by ashort non-coding DNA fragment.

GLV-1j88 was generated by deletion the coding sequence of the A35R genein starting strain GLV-1h73. Thus, in strain GLV-1j88, the vaccinia A35gene is replaced by a short non-coding DNA fragment.

GLV-1j89 was generated by deletion the coding sequence of the A35R genein starting strain GLV-1h74. Thus, in strain GLV-1j89, the vaccinia A35gene is replaced by a short non-coding DNA fragment.

GLV-1h90 was generated by insertion of an expression cassette encodinghuman IL-6 fused to the 3′ end of the cDNA encoding human soluble IL-6receptor (sIL-6R, aa 1-323) under the control of the vaccinia P_(SE)promoter into the HA locus of starting strain GLV-1h68, thereby deletingthe gusA expression cassette at the HA locus of starting GLV-1h68. Thus,in strain GLV-1h90, the vaccinia HA gene is interrupted within thecoding sequence by a DNA fragment containing DNA encoding human IL-6fused to the 3′ end of the cDNA encoding human soluble IL-6 receptoroperably linked to the vaccinia synthetic early promoter.

GLV-1h91 was generated by insertion of an expression cassette encodingsIL-6R under the control of the vaccinia P_(SEL) promoter into the HAlocus of starting strain GLV-1h68, thereby deleting the gusA expressioncassette at the HA locus of starting GLV-1h68. Thus, in strain GLV-1h91,the vaccinia HA gene is interrupted within the coding sequence by a DNAfragment containing DNA encoding human IL-6 fused to the 3′ end of thecDNA encoding human soluble IL-6 receptor operably linked to thevaccinia synthetic early/late promoter.

GLV-1h92 was generated by insertion of an expression cassette encodingsIL-6R under the control of the vaccinia P_(SL) promoter into the HAlocus of starting strain GLV-1h68, thereby deleting the gusA expressioncassette at the HA locus of starting GLV-1h68. Thus, in strain GLV-1h92,the vaccinia HA gene is interrupted within the coding sequence by a DNAfragment containing DNA encoding human IL-6 fused to the 3′ end of thecDNA encoding human soluble IL-6 receptor operably linked to thevaccinia synthetic late promoter.

GLV-1h96 was generated by insertion of an expression cassette encodingthe IL-24 under the control of the vaccinia P_(SE) promoter into theF14.5L locus of starting strain GLV-1h68, thereby deleting the Ruc-GFPfusion gene expression cassette at the F14.5L locus of GLV-1h68. Thus,in strain GLV-1h96, the vaccinia F14.5L gene is interrupted within thecoding sequence by a DNA fragment containing DNA encoding IL-24 operablylinked to the vaccinia synthetic early promoter.

GLV-1h97 was generated by insertion of an expression cassette encodingIL-24 under the control of the vaccinia P_(SEL) promoter into the F14.5Llocus of starting strain GLV-1h68, thereby deleting the Ruc-GFP fusiongene expression cassette at the F14.5L locus of GLV-1h68. Thus, instrain GLV-1h97, the vaccinia F14.5L gene is interrupted within thecoding sequence by a DNA fragment containing DNA encoding FCU operablylinked to the vaccinia synthetic early/late promoter.

GLV-1h98 was generated by insertion of an expression cassette encodingIL-24 under the control of the vaccinia P_(SL) promoter into the F14.5Llocus of starting strain GLV-1h68, thereby deleting the Ruc-GFP fusiongene expression cassette at the F14.5L locus of GLV-1h68. Thus, instrain GLV-1h98, the vaccinia F14.5L gene is interrupted within thecoding sequence by a DNA fragment containing DNA encoding IL-24 operablylinked to the vaccinia synthetic late promoter.

GLV-1h104 was generated by insertion of an expression cassettecontaining DNA encoding truncated human tissue factor fused to theα_(v)β₃-integrin RGD binding motif (tTF-RGD) under the control of thevaccinia synthetic early promoter (P_(SE)) into the TK locus of strainGLV-1h68 thereby deleting the LacZ/rTFr expression cassette at the TKlocus of GLV-1h68. Strain GLV-1h104 retains the Ruc-GFP expressioncassette at the F14.5L locus and the gusA expression cassette at the HAlocus.

GLV-1h105 was generated by insertion of an expression cassettecontaining DNA encoding tTF-RGD fusion protein under the control of thevaccinia synthetic early/late promoter (P_(SEL)) into the TK locus ofstrain GLV-1h68 thereby deleting the LacZ/rTFr expression cassette atthe TK locus of GLV-1h68. Strain GLV-1h105 retains the Ruc-GFPexpression cassette at the F14.5L locus and the gusA expression cassetteat the HA locus.

GLV-1h106 was generated by insertion of an expression cassettecontaining DNA encoding tTF-RGD fusion protein under the control of thevaccinia synthetic late promoter (P_(SL)) into the TK locus of strainGLV-1h68 thereby deleting the LacZ/rTFr expression cassette at the TKlocus of GLV-1h68. Strain GLV-1h106 retains the Ruc-GFP expressioncassette at the F14.5L locus and the gusA expression cassette at the HAlocus.

GLV-1h107 was generated by insertion of an expression cassettecontaining DNA encoding scFv anti-VEGF-FLAG fusion protein (G6-FLAG)under the control of the vaccinia synthetic early promoter (P_(SE)) intothe TK locus of strain GLV-1h68 thereby deleting the LacZ/rTFrexpression cassette at the TK locus of GLV-1h68. Strain GLV-1h107retains the Ruc-GFP expression cassette at the F14.5L locus and the gusAexpression cassette at the HA locus.

GLV-1h108 was generated by insertion of an expression cassettecontaining DNA encoding G6-FLAG fusion protein under the control of thevaccinia synthetic early/late promoter (P_(SEL)) into the TK locus ofstrain GLV-1h68 thereby deleting the LacZ/rTFr expression cassette atthe TK locus of GLV-1h68. Strain GLV-1h108 retains the Ruc-GFPexpression cassette at the F14.5L locus and the gusA expression cassetteat the HA locus.

GLV-1h109 was generated by insertion of an expression cassettecontaining DNA encoding G6-FLAG fusion protein under the control of thevaccinia synthetic late promoter (P_(SL)) into the TK locus of strainGLV-1h68 thereby deleting the LacZ/rTFr expression cassette at the TKlocus of GLV-1h68. Strain GLV-1h109 retains the Ruc-GFP expressioncassette at the F14.5L locus and the gusA expression cassette at the HAlocus.

2. VV Transfer Vectors Employed for the Production of Modified VacciniaViruses

The following vectors were constructed and employed as described belowto generate the recombinant vaccinia viral strains.

a. pNCVVhaT: For Insertion of Non-Coding Heterologous DNA into theVaccinia Virus HA Locus

The pNCVVhaT vector (SEQ ID NO: 4) was employed to create vaccinia virusstrains GLV-1h70 and GLV-1h86 having the following genotypes: F14.5L:(P_(SEL))Ruc-GFP, TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ (strain GLV-1h70),HA: HindIII-BamHI and F14.5L: (P_(SEL))Ruc-GFP, TK: SacI-BamHI, HA:HindIII-BamHI (strain GLV-1h86). Strains GLV-1h70 and GLV-1h86 weregenerated by inserting a short non-coding DNA fragment containingHindIII and BamHI sites (SEQ ID NO: 5; taagcttcgcaggatccc) into the HAlocus of strains GLV-1h68 and GLV-1h72, respectively, thereby deletingthe gusA expression cassette at the hemagglutinin (HA) locus of GLV-1h68and GLV-1h72. Vector pNCVVhaT contains the non-coding DNA fragmentflanked by sequences of the HA gene, the E. coli guaninephosphoribosyltransferase (gpt) gene under the control of the vacciniavirus P_(7.5kE) promoter for transient dominant selection of virus thathas incorporated the vector, and sequences of the pUC plasmid. The leftand right flanking sequences of the W HA gene (also named A56R, seenucleotides 161420 to 162352 of SEQ ID NO: 2) that were incorporatedinto the vector correspond to nucleotides 161423 to 161923 andnucleotides 162037 to 162394, respectively of SEQ ID NO: 2. The HA geneflanking DNAs were PCR-amplified from W LIVP using Platinum PCR SuperMixHigh Fidelity (Invitrogen, Carlsbad, Calif.) and the following primerscontaining the non-coding DNA sequence:

(SEQ ID NO: 6) Left flank: 5′-GCGCATATGACACGATTACCAATACTTTTG-3′ and (SEQID NO: 7) 5′-GTCGGGATCCTGCGAAGCTTAGATTTCGAATACCGACGAGC-3′, Right Flank:(SEQ ID NO: 8) 5′-GAAATCTAAGCTTCGCAGGATCCCGACTCCGGAACCAATTACTG-3′ and(SEQ ID NO: 9) 5′-GCGGAATTCTGATAGATTTTACTATCCCAG-3′.The two fragments were joined using the method of gene-splicing byoverlapping extension (see, e.g., Horton et al., Methods Enzymol.,217:270-279 (1993)). The resulting fragment was digested with NdeI andEcoRI and cloned into the same-cut vector pUCP7.5-gpt-1 (SEQ ID NO: 10)to generate the construct pNCVVhaT. The flanking sequences of HA in thetarget vector were confirmed by sequencing and were identical tonucleotides 161423 to 161923 of SEQ ID NO: 2 (left flank) andnucleotides 162037 to 162394 of SEQ ID NO: 2 (right flank).

b. pNCVVf14.51T: For Insertion of Non-Coding Heterologous DNA into theVaccinia F14.5L Locus

The pNCVVf14.51T vector (SEQ ID NO: 11) was employed to create vacciniavirus strains GLV-1h71, GLV-1h73 and GLV-1h85 having the followinggenotypes: F14.5L: BamHI-HindIII, TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ(GLV-1h71), HA: (P_(11k))gusA; F14.5L: BamHI-HindIII, TK:(P_(SEL))rTrfR-(P7.5 k)LacZ (GLV-1h73), HA:HindIII-BamHI and F14.5L:BamHI-HindIII, TK:SacI-BamHI (GLV-1h85), HA: (P_(11k))gusA. StrainsGLV-1h71, GLV-1h73 and GLV-1h85 were generated by inserting a shortnon-coding DNA fragment containing Bam HI and HindIII sites (SEQ ID NO:12; aggatcctgcgaagct) into the F14.5L locus of strains GLV-1h68,GLV-1h70 and GLV-h72, respectively, thereby deleting the Ruc-GFP fusiongene expression cassette at the F14.5L locus of these strains. VectorpNCVVf14.51T contains the non-coding DNA fragment flanked by sequencesof the F14.5L gene, the E. coli guanine phosphoribosyltransferase (gpt)gene under the control of the vaccinia virus P_(7.5kE) promoter fortransient dominant selection of virus that has incorporated the vector,and sequences of the pUC plasmid. The left and right flanking sequencesof the VV F14.5L gene (see nucleotides 41476 to 41625 of SEQ ID NO: 2)that were incorporated into the vector correspond to nucleotides 41593to 42125 and nucleotides 41018 to 41592, respectively of SEQ ID NO: 2.The F14.5L gene flanking DNAs were PCR-amplified from VV LIVP usingPlatinum PCR SuperMix High Fidelity and the following primers containingthe non-coding DNA sequence:

(SEQ ID NO: 13) Left Flank: 5′-GCGCATATGTAGAAGAATTGATAAATATG-3′ and (SEQID NO: 14) 5′-GCCGCAGGATCCTGCGAAGCTTACAGACACGAATATGACTAAACCGA TG-3′,(SEQ ID NO: 15) Right Flank:5′-GTCTGTAAGCTTCGCAGGATCCTGCGGCCGCCATCGTCGGTGTGTTG TC-3′ and (SEQ ID NO:16) 5′-GCGGAATTCAGAGGATTACAACAAAAAGATG-3′.The two fragments were joined together as described above (gene-splicingby overlapping extension). The resulting fragment was digested with NdeIand EcoRI and cloned into the same-cut vector pUCP7.5-gpt-1 (SEQ ID NO:10) to generate the construct pNCVVf14.51T (SEQ ID NO: 11). The flankingsequences of F14.5L in the target vector were confirmed by sequencingand were identical to nucleotides 41593 to 42125 of SEQ ID NO: 2 (leftflank) and nucleotides 41018 to 41592 of SEQ ID NO: 2 (right flank).

c. pCR-TKLR-gpt2: For Insertion of Non-Coding Heterologous DNA in theVaccinia TK Locus

The pCR-TKLR-gpt2 vector (SEQ ID NO: 17) was employed to create vacciniavirus strains GLV-1h72 and GLV-1h74 having the following genotypes:F14.5L: (P_(SEL))Ruc-GFP, TK: SacI-BamHI (GLV-1h72), HA: (P_(11k))gusAand F14.5L: BamHI-HindIII, TK: SacI-BamHI (GLV-1h74), HA:HindIII-BamHI.Strain GLV-1 h72 was generated by inserting a short non-coding DNAfragment containing SacI and BamHI sites (SEQ ID NO: 18;ggtaccgagctcggatcc) into the TK locus of starting strain GLV-1h68thereby deleting the LacZ/rTFr expression cassette at the TK locus ofGLV-1h68. Strain GLV-1h74 was generated by inserting the shortnon-coding DNA fragment containing SacI and Bam HI sites into the TKlocus of strain GLV-1 h73 thereby deleting the LacZ/rTFr expressioncassette at the TK locus of GLV-1 h73.

Vector pCR-TKLR-gpt2 was generated from vector pCR2.1 (Invitrogen,Carlsbad, Calif., SEQ ID NO: 21) and contains the non-coding DNAfragment flanked by sequences of the TK gene and the E. coli guaninephosphoribosyltransferase (gpt) gene under the control of the vacciniavirus P_(7.5kE) promoter for transient dominant selection of virus thathas incorporated the vector. The left flank (TK_(L)) of the TK locus inthe LIVP genome that was incorporated into the vector corresponds tonucleotides 79726 to 80231 of SEQ ID NO: 2 (TK locus in the LIVP genomeis located at nucleotides 78142 to 80961 of SEQ ID NO: 2). The leftflank DNA was PCR amplified with the primers TK_(L)-5(5′-ATAAGCTTTGTTACAGATGGAAGGGTCAAA-3′, SEQ ID NO: 19) and TK_(L)-3(5′-AGGTACCGTTTGCCATACGCTCACAGA-3′, SEQ ID NO: 20) using Invitrogen HighFidelity PCR mix. The PCR product was digested with HindIII and KpnI,and inserted into the corresponding sites in vector pCR2.1 (Invitrogen,Carlsbad, Calif., SEQ ID NO: 21), resulting in pCP-TKL1 (SEQ ID NO: 22).The right flanking region (TK_(R)) of the TK locus in the LIVP genomethat was incorporated into the vector corresponds to nucleotides 80211to 80730 of SEQ ID NO: 2. The right flank DNA was PCR amplified with theprimers: TK_(R)-5 (5′-TGAGCTCGGATCCTTCTGTGAGCGTATGGCAAA-3′, SEQ ID NO:23) and TK_(R)-3 (5′-TTACTAGTACACTACGGTGGCACCATCT-3′, SEQ ID NO: 24).The PCR product was digested with BamHI and SpeI and cloned into thecorresponding sites in vector pCR2.1 to yield pCR-TKR4 (SEQ ID NO: 25).The pCR-TKL1 and pCR-TKR4 contained the correct sequences of TK_(L) andTK_(R), respectively, as confirmed by sequencing and were identical tonucleotides 79726 to 80231 of SEQ ID NO: 2 (left flank) and nucleotides80211 to 80730 of SEQ ID NO: 2 (right flank). The insert TK_(L) was thenexcised from pCR-TKL1 by restriction digestion with HindIII and BamHIand inserted into the same-cut vector pCR-TKR4 to yield pCR-TKLR1 (SEQID NO: 26) thereby joining the left and right flanking sequences withthe non-coding DNA between them in a single fragment.

In order to add DNA encoding Escherichia coli guaninephosphoribosyltransferase (gpt) linked to the vaccinia virus promoterp7.5 k to pCR-TKLR1 for use in transient dominant selection, a DNAfragment containing these elements was amplified with the primers gpt5(5′-TCCCAGTCACGACGTTGTAA-3′, SEQ ID NO: 27) and gpt3(5′-TGATTACGCCAAGCTGATCC-3′, SEQ ID NO: 28) from pUCP7.5-gpt-1 andcloned into vector pCR2.1. The sequence of the insert p7.5 k-gpt wasconfirmed and released with EcoRI and cloned into the same-cut vectorpCR-TKLR1 to generate the final transfer vector pCR-TKLR-gpt2 (SEQ IDNO: 17).

d. pNCVVhaT-SEL-hk5: For Insertion of an Expression Cassette EncodingPlasminogen Kringle 5 Domain Under the Control of the Vaccinia P_(SEL)Promoter into the Vaccinia HA Locus

Vector pNCVVhaT-SEL-hk5 (SEQ ID NO: 41) was employed to develop strainGLV-1h81 having the following genotype: F14.5L: (P_(SEL))Ruc-GFP, TK:(P_(SEL))rTrfT-(P_(7.5k))LacZ, HA: (P_(SEL))hk-5. Strain GLV-1h81 wasgenerated by inserting DNA encoding the human plasminogen kringle 5domain (SEQ ID NO: 42) operably linked to the vaccinia virus syntheticearly/late promoter (P_(SEL)) (SEQ ID NO: 29) into the HA locus ofstarting strain GLV-1h68 thereby deleting the gusA expression cassetteat the HA locus of GLV-1h68. Vector pNCVVhaT-SEL-hk5 contains a DNAfragment encoding the human plasminogen kringle 5 domain operably linkedto the vaccinia synthetic early/late promoter (P_(SEL)), sequences ofthe HA gene flanking the (P_(SEL))hk-5 DNA fragment, the E. coli guaninephosphoribosyltransferase (gpt) gene under the control of the vacciniavirus P_(7.5kE) promoter for transient dominant selection of virus thathas incorporated the vector, and sequences of the pUC plasmid.

To generate vector pNCVVhaT-SEL-hk5, DNA encoding human plasminogenkringle 5 was PCR-amplified from the plasmid pBLAST-hKringle5(Invivogen, San Diego, Calif.; SEQ ID NO: 43) using AccuPrime PfxSuperMix (Invitrogen, Carlsbad, Calif.) and primers:5′-GCGAAGCTTACCATGTACAGGATGCAACTCCTGTCTTG-3′ (SEQ ID NO: 44) and5′-GCGGGATCCAGAAAAACTAATCAAATGAAGGGGCCGCACACTG-3′ (SEQ ID NO: 45). ThePCR product was digested with HindIII and BamHI and cloned into thesame-cut vector pNCVVhaT-SEL-ADP-V5 (SEQ ID NO: 46); similar topNCVVhaT, but contains ADP-V5 under the control of the syntheticearly/late promoter in between the flanking sequences of HA to replaceadenovirus death protein (ADP) gene tagged with V5 at 3′ end. Thesequence of the human plasminogen kringle 5 domain was confirmed bysequencing.

e. pNCVVhaT-ftn: for Insertion of an Expression Cassette Encoding E.coli Ferritin Under the Control of the Vaccinia P_(SEL) Promoter intothe Vaccinia HA Locus

Vector pNCVVhaT-ftn (SEQ ID NO: 47) was employed to develop strainsGLV-1h82 and GLV-1h83 having the following genotypes:, F14.5L:(P_(SEL))Ruc-GFP, TK: (P_(SEL))TrfR-(P7.5 k)LacZ (strain GLV-1h82), HA:(P_(SEL))ftn, and F14.5L: (P_(SEL))Ruc-GFP, TK: (P_(SEL))rTrfR-(P7.5k)LacZ (strain GLV-1h83), HA: (P_(SEL))ftn. Strains GLV-1h82 andGLV-1h83 were generated by inserting DNA encoding E. coli ferritin (ftn)(SEQ ID NO: 48) operably linked to the vaccinia virus syntheticearly/late promoter (P_(SEL)) (SEQ ID NO: 29) into the HA locus ofstarting strains GLV-1h22 and GLV-1h68, respectively, thereby deletingthe gusA expression cassette at the HA locus of these starting strains.Vector pNCVVhaT-ftn contains a DNA fragment encoding E. coli ferritinoperably linked to the vaccinia synthetic early/late promoter (P_(SEL)),sequences of the HA gene flanking the (P_(SEL))ftn DNA fragment, the E.coli guanine phosphoribosyltransferase (gpt) gene under the control ofthe vaccinia virus P_(7.5kE) promoter for transient dominant selectionof virus that has incorporated the vector, and sequences of the pUCplasmid.

To generate vector pNCVVhaT-ftn, DNA encoding E. coli ferritin (ftn) wasamplified from genomic DNA of E. coli Top10 (Invitrogen, Carlsbad,Calif.) using the following primers:

(SEQ ID NO: 49) 5′SSEL-ftn-VV3(5′-AAAGATAAGCTTAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATACCATGCTGAAACCAGAAATGATTGAA-3′) and (SEQ ID NO: 50) 3′ftn-VV2(5′-ATAATAGGATCCTTAGTTTTGTGTGTCGAGGGT-3′).

Primer 5′SSEL-ftn-VV3 introduces a HindIII site, the P_(SEL) promotersequence for vaccinia virus synthetic strong early/late expression, anda Kozak sequence (ACC) in front of the start codon of ftn. 3′ftn-VV2introduces a BamHI restriction site. The PCR product as well as theplasmid pNCVVhaT (SEQ ID NO: 4) were digested with BamHI and HindIII,ligated, and transformed into E. coli Top10 to yield pNCVVhaT-ftn (SEQID NO: 47). This final cloning step places the (P_(SEL))ftn expressioncassette between the left and right HA gene flanking sequences inpNCVVhaT and eliminates the non-coding DNA that is located between theseflanking sequences in pNCVVhaT.

f. pCR-TK-SEL-mRFP1: for Insertion of an Expression Cassette Encoding aFusion Protein of CBG99 and mRFP1 Under the Control of the VacciniaP_(SEL) Promoter into the Vaccinia TK Locus

Vector pCR-TK-SEL-mRFP1 (SEQ ID NO: 51) was employed to develop strainGLV-1h84 having the following genotype: F14.5L: BamHI-HindIII, TK:(P_(SEL))CBG99-mRFP1, HA: HindIII-BamHI. Strain GLV-1h84 was generatedby inserting DNA encoding a fusion protein of CBG99 (green-emittingclick beetle luciferase) and mRFP1 (red fluorescent protein) linkedthrough a picornavirus 2A element (SEQ ID NO: 52) operably linked to thevaccinia virus synthetic early/late promoter (P_(SEL)) (SEQ ID NO: 29)into the TK locus of strain GLV-1h73 thereby deleting the rTrfR-LacZexpression cassette at the TK locus of strain GLV-1h73. VectorpCR-TK-SEL-mRFP I contains a DNA fragment encoding a CBG99-mRFP I fusionprotein operably linked to the vaccinia synthetic early/late promoter(P_(SEL)), sequences of the TK gene flanking the (P_(SEL))-fusionprotein-encoding DNA fragment, the E. coli guaninephosphoribosyltransferase (gpt) gene under the control of the vacciniavirus P7.5 k early and late promoter for transient dominant selection ofvirus that has incorporated the vector, and sequences of the pUCplasmid.

To generate vector pCR-TK-SEL-mRFP1, cDNA encoding the fusion proteinCBG99 (green-emitting click beetle luciferase) and mRFP1 (redfluorescent protein) linked through the picornavirus 2A element was PCRamplified from CBG99-2A-mRFP1 (SEQ ID NO: 53) with the primers:

(SEQ ID NO: 54) mRFP5 (5′-GTCGACGCCACCATGGTGAAGCGTGAG-3′) and (SEQ IDNO: 55) mRFP3 (5′-TCATTAGGCGCCGGTGGAGT-3′).

The PCR product was cloned into vector pCR-Blunt II-TOPO (Invitrogen;SEQ ID NO: 40) to yield pCRII-mRFP (SEQ ID NO: 56). After confirming thesequence, the CBG99-mRFP1 fusion cDNA molecule (SEQ ID NO: 52) wasreleased by SalI and EcoRV restriction enzyme digest and inserted intopCR-SEL4 (SEQ ID NO: 33), precut with SalI and SmaI to generate plasmidpCR-SEL-mRFP1 (SEQ ID NO: 57). (pCR-SEL4 was constructed as follows: ThecDNA spanning the synthetic early/late promoter P_(SEL) (SEQ ID NO: 29)for vaccinia virus and the multiple cloning site (MCS) region in pSC65(SEQ ID NO: 30) was PCR amplified with the primers SEL5(5′-TAGAGCTCGGTTTGGAATTAGTGAAAGC-3′) (SEQ ID NO: 31) and SEL3(5′-TAGAGCTCTCCAGACATTGTTGAATTAG-3′) (SEQ ID NO: 32), and cloned intothe TA cloning site of vector pCR2.1 to yield pCR-SEL4 (SEQ ID NO: 33)).This intermediate cloning step placed the fusion cDNA molecule under thecontrol of vaccinia virus synthetic early/late promoter (P_(SEL)). TheSEL-CBG99-mRFP1 expression cassette was then released by SacI digestionand cloned into the same-cut vaccinia virus TK locus transfer vectorpCR-TKLR-gpt2 (SEQ ID NO: 17) to give the final constructpCR-TK-SEL-mRFP1 (SEQ ID NO: 51). This final cloning step placed the(P_(SEL))CBG99-mRFP1 expression cassette between the left and right TKgene flanking sequences in pCR-TKLR-gpt2 and eliminated the non-codingDNA that is located between these flanking sequences in pCR-TKLR-gpt2.

g. pCR-gpt-dA35R6: For Deletion of the A35R Locus and Insertion of aNon-Coding Heterologous DNA with Multiple Cloning Sites

Vector pCR-gpt-dA35R-6 (SEQ ID NO: 89) was employed to create vacciniastrains GLV-1j87, GLV-1j88 and GLV-1j89, having the following genotypes:F14.5L: (P_(SEL))Ruc-GFP, TK: (P_(SEL))rTrfR-(P7.5 k)LacZ, HA:(P_(11k))gusA, A35R: deleted, multiple cloning sites (MCS) (strainGLV-1j87); F14.5L: BamHI-HindIII, TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ, HA:HindIII-BamHI, A35R: deleted, MCS (strain GLV-1j88); and F14.5L:BamHI-HindIII, TK: SacI-BamHI, HA: HindIII-BamHI, A35R: deleted, MCS(strain GLV-1j89). Strains GLV-1j87, GLV-1j88 and GLV-1j89, weregenerated by inserting a short DNA fragment with multiple cloning sites(HindIII, SacI and BamHI) into the A35R locus of strains GLV-1h68,GLV-1h73 and GLV-1 h74, respectively, thereby creating a fusion of theflanking A34R and A36R regions and deleting the A35R gene. VectorpCR-gpt-dA35R-6 contains a non-coding DNA fragment with multiple cloningsites flanked by sequences that flank the A35R gene (a fusion of A34Rand A36R regions) and the E. coli guanine phosphoribosyltransferase(gpt) gene under the control of the vaccinia virus P_(7.5kE) promoterfor transient dominant selection of virus that has incorporated thevector.

The left and right flanking sequences of A35R, the A34R and A36Rregions, were PCR amplified. The A34R gene region was PCR amplified withprimers

A34R-L: 5′-ATCTCGAGTGAGGATACATGGGGATCTGATG-3′ (SEQ ID NO: 66) and

A34R-R: 5′-ATGAGCTCCCGGGAAGCTTGGCGGCGTACGTTAACGAC-3′ (SEQ ID NO: 67),using LIVP genomic DNA (SEQ ID NO: 2) as the template.

The A36R gene region was PCR amplified with primers

A36R-L: 5′-ATGAGCTCGGATCCTGCATATCAGACGGCAATGG-3′ (SEQ ID NO: 68) and

A36R-R: 5′-ATGGGCCCATCGCTATGTGCTCGTCTA-3′ (SEQ ID NO: 69), using LIVPgenomic DNA (SEQ ID NO: 2) as the template.

The A34R and A36R PCR products were digested with SacI, and therestricted products were then purified and ligated together. The A34Rand A36R ligation product was used as the template for PCR amplificationof the A34R and A36R fusion cDNA, with primers A34R-L and A36R-R. Theamplified fusion cDNA was cloned into pCR-Blunt II-TOPO vector(Invitrogen; SEQ ID NO: 40) to generate vector pCRII-dA35R-1 (SEQ ID NO:87). The resulting vector was confirmed by sequencing.

A p7.5-gpt expression vector with the HindIII, SacI and BamHI sitesremoved was then generated. The TK region in the TK locus transfervector pCR-TKLR-gpt2 (SEQ ID NO: 17) was removed with HindIII and SpeIdigestion. The vector fragment was blunt ended with Klenow treatment,and then ligated to generate construct pCR-dTK-gpt1 (SEQ ID NO: 88). Therestriction sites HindIII, SacI and BamHI are removed in the resultingpCR-dTK-gpt1 vector (SEQ ID NO: 88).

To generate pCR-gpt-dA35R-6, the A34R and A36R fusion cDNA was releasedfrom pCRII-dA35R-1 (SEQ ID NO: 87) by XhoI and ApaI digestion, andinserted into vector pCR-dTK-gpt1 (SEQ ID NO: 88), precut with XhoI andApaI. The resulting construct pCR-gpt-dA35R-6 (SEQ ID NO: 89) wasconfirmed by sequencing.

h. HA-SE-IL-6-1: For Insertion of an Expression Cassette EncodingsIL-6R/IL-6 Under the Control of the Vaccinia P_(SE) Promoter into theVaccinia HA Locus.

Vector HA-SE-IL-6-1 (SEQ ID NO: 77) was employed to develop strainGLV-1h90 having the following genotype: F14.5L: (P_(SEL))RUC-GFP, TK:(P_(SEL))rTrfR-(P_(7.5k))LacZ, HA: (P_(SE))sIL-6R/IL-6. Strain GLV-1h90was generated by inserting DNA encoding a fusion protein of human IL-6(encoding amino acids 29-212) fused to the human soluble IL-6 receptor(sIL-6R) (amino acids 1-323) by a linker sequence (encodingRGGGGSGGGGSVE (SEQ ID NO: 90); complete sequence of sIL-6R/IL insert(SEQ ID NO: 106)) operably linked to the vaccinia virus synthetic earlypromoter (P_(SE)) (SEQ ID NO: 35) into the HA locus of strain GLV-1h68,thereby deleting the gusA expression cassette at the HA locus ofstarting GLV-1h68. Vector HA-SE-IL-6-1 contains a DNA fragment encodingthe sIL-6R/IL-6 fusion protein operably linked to the vaccinia syntheticearly promoter (P_(SE)) and sequences of the HA gene flanking the(P_(SE))-fusion protein-encoding DNA fragment.

Plasmid pCR-SE1 (SEQ ID NO: 36), containing the vaccinia synthetic earlypromoter, i.e., P_(SE), was used as the source of the vaccinia syntheticearly promoter in generating vector HA-SE-IL-6-1. pCR-SE1 wasconstructed as follows. The multiple cloning site (MCS) region in pSC65(Moss and Earl, Current Protocols in Molecular Biology, 16.17.4, 1998;SEQ ID NO: 30) was PCR amplified with the primers:

SE5: (SEQ ID NO: 34) 5′-TAGAGCTCAAAAATTGAAAAACTAGCGTCTTTTTTTGCTCGAAGTCGACAGATCTAGGCCTG-3′, (SEQ ID NO: 35) containing the sequence forsynthetic early promoter P_(SE), and SEL3: (SEQ ID NO: 32)5′-TAGAGCTCTCCAGACATTGTTGAATTAG-3′.The resulting PCR product was inserted into the TA cloning site ofvector pCR2.1 to obtain pCR-SE1 (SEQ ID NO: 36).

To generate vector HA-SE-IL-6-1, cDNA encoding the fusion proteinsIL-6R/IL-6 was PCR amplified from pCDM8-H-IL-6 (U.S. Pat. No.7,112,436) with the primers:

(SEQ ID NO: 62) 5′-GTCGACCCACCATGCTGGCCGTCGGCTGCGC-3′ and (SEQ ID NO:63) 5′-GGTACCCTAGAGTCGCGGCCGCGACC-3′.

The PCR product was cloned into vector pCR-Blunt 1′-TOPO (Invitrogen;SEQ ID NO: 40) to yield pCRII-IL6-3 (SEQ ID NO: 73). After confirmingthe sequence, the sIL-6R/IL-6 fusion cDNA molecule (SEQ ID NO: 106) wasreleased by KpnI (blunt ended) and SalI restriction enzyme digest andinserted into vector pCR-SE1 (SEQ ID NO: 36), precut with SalI and SmaIto generate plasmid pCR-SE-IL6-7 (SEQ ID NO: 74), thus placing the IL-6fusion cDNA under the control of vaccinia virus synthetic early (SE)promoter.

The cDNA of SE-IL6 was released from pCR-SE-IL6-7 (SEQ ID NO: 74) byHindIII and BamHI restriction enzyme digest and inserted into the HAtransfer vector, pNCVVhaT (SEQ ID NO: 4), precut with HindIII and BamHIto generate plasmid HA-SE-IL6-1 (SEQ ID NO: 77). The SL-sIL-6R/IL-6fusion expression was confirmed by sequencing.

i. HA-SEL-IL-6-1: For Insertion of an Expression Cassette EncodingsIL-6R/IL-6 Under the Control of the Vaccinia P_(SEL) Promoter into theVaccinia HA Locus.

Vector HA-SEL-IL-6-1 (SEQ ID NO: 79) was employed to develop strainGLV-1h91 having the following genotype: F14.5L: (P_(SEL))Ruc-GFP, TK:(P_(SEL))rTrfR-(P_(7.5k))LacZ, HA: (P_(SEL))sIL-6R/IL-6. Strain GLV-1h91was generated by inserting DNA encoding the sIL-6R/IL-6 fusion proteinoperably linked to the vaccinia virus synthetic early/late promoter(P_(SEL)) (SEQ ID NO: 29) into the HA locus of starting strain GLV-1h68,thereby deleting the gusA expression cassette at the HA locus ofstarting GLV-1h68. Vector HA-SL-IL-6-1 contains a DNA fragment encodingthe sIL-6R/IL-6 fusion protein operably linked to the vaccinia syntheticearly promoter (P_(SEL)) and sequences of the HA gene flanking the(P_(SEL))-fusion protein-encoding DNA fragment.

Plasmid pCR-SEL4 (SEQ ID NO: 33; see (f) above for construction ofpCR-SEL4), containing the vaccinia synthetic early/late promoter, i.e.,P_(SEL), was used as the source of the vaccinia synthetic early/late ingenerating vector HA-SEL-IL-6-1.

To generate vector HA-SL-IL-6-1, the sIL-6R/IL-6 fusion cDNA molecule(SEQ ID NO: 106) was released from vector pCRII-IL6-3 (see (h) above;SEQ ID NO: 73) by KpnI and SalI restriction enzyme digest and insertedinto vector pCR-SEL4 (SEQ ID NO: 33), precut with SalI and SmaI togenerate plasmid pCR-SEL-IL6-2 (SEQ ID NO: 76), thus placing the IL-6fusion cDNA under the control of vaccinia virus synthetic early/late(P_(SEL)) promoter.

The cDNA of SEL-IL6 was released from pCR-SEL-IL6-2 (SEQ ID NO: 76) byHindIII restriction enzyme digest and inserted into the HA transfervector, pNCVVhaT (SEQ ID NO: 4), precut with HindIII to generate plasmidHA-SEL-IL6-1 (SEQ ID NO: 79). The SEL-sIL-6R/IL-6 fusion expressioncassette was confirmed by sequencing.

j. HA-SL-IL-6-1: For Insertion of an Expression Cassette EncodingsIL-6R/IL-6 Under the Control of the Vaccinia P_(SL) Promoter into theVaccinia HA Locus.

Vector HA-SL-IL-6-1 (SEQ ID NO: 78) was employed to develop strainGLV-1h92 having the following genotype: F14.5L: (P_(SEL))RUC-GFP, TK:(P_(SEL))rTrfR-(P_(7.5k))LacZ, HA: (P_(SL))sIL-6R/IL-6. Strain GLV-1h92was generated by inserting DNA encoding the sIL-6R/IL-6 fusion protein(SEQ ID NO: 106) operably linked to the vaccinia virus synthetic latepromoter (P_(SL)) (SEQ ID NO: 38) into the HA locus of starting strainGLV-1h68, thereby deleting the gusA expression cassette at the HA locusof starting GLV-1h68. Vector HA-SL-IL-6-1 contains a DNA fragmentencoding the sIL-6R/IL-6 fusion protein operably linked to the vacciniasynthetic late promoter (P_(SL)) and sequences of the HA gene flankingthe (P_(SL))-fusion protein-encoding DNA fragment.

Plasmid pCR-SL3 (SEQ ID NO: 39), containing the vaccinia synthetic latepromoter, i.e., P_(SL), was used as the source of the vaccinia syntheticlate promoter in generating vector HA-SL-IL-6-1 (SEQ ID NO: 78). Toconstruct pCR-SL3, the MCS region in pSC65 was PCR amplified with theprimers:

SL5: (SEQ ID NO: 37) 5′-TAGAGCTCTTTTTTTTTTTTTTTTTTTTGGCATATAAATAAGTCGACAGATCTAGGCCTG-3′, (SEQ ID NO: 38) containing the sequence for syntheticlate promoter P_(SL), and SEL3: (SEQ ID NO: 32)5′-TAGAGCTCTCCAGACATTGTTGAATTAG-3′).The resulting PCR product was cloned into the TA cloning site of vectorpCR2.1 to yield pCR-SL3 (SEQ ID NO: 39).

To generate vector HA-SL-IL-6-1, the sIL-6R/IL-6 fusion cDNA molecule(SEQ ID NO: 106) was released from vector pCRII-IL6-3 (see (h) above;SEQ ID NO: 73) by KpnI and SalI restriction enzyme digest and insertedinto vector pCR-SL3 (SEQ ID NO: 39), precut with SalI and SmaI togenerate plasmid pCR-SL-IL6-2 (SEQ ID NO: 75), thus placing the IL-6fusion cDNA under the control of vaccinia virus synthetic late (SL)promoter.

The cDNA of SL-sIL-6R/IL-6 was released from pCR-SL-IL6-2 (SEQ ID NO:75) by HindIII and BamHI restriction enzyme digest and inserted into theHA transfer vector, pNCVVhaT (SEQ ID NO: 4), precut with HindIII andBamHI to generate plasmid HA-SL-IL6-1 (SEQ ID NO: 78). TheSL-sIL-6R/IL-6 fusion expression cassette was confirmed by sequencing.

k. FSE-IL-24: For Insertion of an Expression Cassette Encoding IL-24Under the Control of the Vaccinia P_(SE) Promoter into the VacciniaF14.5L Locus.

Vector FSE-IL-24 (SEQ ID NO: 84) was employed to develop strain GLV-1h96having the following genotype: F14.5L: (P_(SE))IL-24, TK:(P_(SEL))rTrfR-(P_(7.5k))LacZ, HA: (P_(11k))gusA. Strain GLV-1h96 wasgenerated by inserting DNA encoding human IL-24 operably linked to thevaccinia virus synthetic early promoter (P_(SE)) (SEQ ID NO: 35) intothe F14.5L locus of strain GLV-1h68, thereby deleting the Ruc-GFP fusiongene expression cassette at the F14.5L locus of GLV-1h68. VectorFSE-IL-24 contains a DNA fragment encoding the IL-24 protein operablylinked to the vaccinia synthetic early promoter (P_(SE)) and sequencesof the F14.5L gene flanking the (P_(SE))-fusion protein-encoding DNAfragment.

Plasmid pCR-SE1 (SEQ ID NO: 36; see (h) above for description ofpCR-SE1), containing the vaccinia synthetic early promoter, i.e.,P_(SE), was used as the source of the vaccinia synthetic early promoterin generating vector FSE-IL-24.

To generate vector FSE-IL-24, cDNA encoding the human IL-24 was PCRamplified from cDNA clone MGC:8926 (complete cds from Origene Trueclonecollection) with the primers:

(SEQ ID NO: 64) 5′- GTCGACCACCATGAATTTTCAACAGAGGCTGC-3′ and (SEQ ID NO:65) 5′- CCCGGGTTATCAGAGCTTGTAGAATTTCTGCATC-3′.

The PCR product was cloned into vector pCR-Blunt II-TOPO (Invitrogen;SEQ ID NO: 40) to yield pCRII-IL24-3 (SEQ ID NO: 80). After confirmingthe sequence, the IL-24 cDNA molecule (SEQ ID NO: 107) was released bySalI and SmaI digestion and inserted into vector pCR-SE1 (SEQ ID NO:36), precut with SalI and SmaI to generate plasmid pCR-SE-IL24-2 (SEQ IDNO: 81), thus placing the IL-24 cDNA under the control of vaccinia virussynthetic early (SE) promoter.

The cDNA of SE-IL24 was released from pCR-SE-IL24-2 (SEQ ID NO: 81) byHindIII and BamHI restriction enzyme digest and inserted into the F14.5Ltransfer vector, pNCVVf14.51T (SEQ ID NO: 11), precut with HindIII andBamHI to generate plasmid FSE-IL24-1 (SEQ ID NO: 84). The SL-IL-24expression was confirmed by sequencing.

l. FSEL-IL-24: For Insertion of an Expression Cassette Encoding IL-24Under the Control of the Vaccinia P_(SEL) Promoter into the VacciniaF14.5L Locus.

Vector FSEL-IL24-1 (SEQ ID NO: 86) was employed to develop strain GLV-1h97 having the following genotype: F14.5L: (P_(SEL))IL-24, TK:(P_(SEL))rTrfR-(P_(7.5k))LacZ, HA: (P_(11k))gusA. Strain GLV-1h97 wasgenerated by inserting DNA human IL-24 operably linked to the vacciniavirus synthetic early/late promoter (P_(SEL)) (SEQ ID NO: 29) into theF14.5L locus of strain GLV-1h68, thereby deleting the Ruc-GFP fusiongene expression cassette at the F14.5L locus of GLV-1h68. VectorFSEL-IL24-1 contains a DNA fragment encoding the IL-24 protein operablylinked to the vaccinia synthetic early/late promoter (P_(SEL)),sequences of the F14.5L gene flanking the (P_(SEL))-fusionprotein-encoding DNA fragment, the E. coli guaninephosphoribosyltransferase (gpt) gene under the control of the vacciniavirus P7.5 k early and late promoter for transient dominant selection ofvirus that has incorporated the vector, and sequences of the pUCplasmid.

Plasmid pCR-SEL4 (SEQ ID NO: 33; see (f) above for construction ofpCR-SEL4), containing the vaccinia synthetic early/late promoter, i.e.,P_(SEL), was used as the source of the vaccinia synthetic early/late ingenerating vector FSEL-IL24-1.

To generate vector FSEL-IL24-1, the IL-24 cDNA molecule (SEQ ID NO: 107)was released from vector pCRII-IL24-3 (see (n) above; SEQ ID NO: 80) byKpnI and SalI restriction enzyme digest and inserted into vectorpCR-SEL4 (SEQ ID NO: 33), precut with SalI and SmaI to generate plasmidpCR-SEL-IL24-2 (SEQ ID NO: 83), thus placing the IL-6 fusion cDNA underthe control of vaccinia virus synthetic early/late (P_(SEL)) promoter.

The cDNA of SEL-IL24 was released from pCR-SEL-IL24-2 (SEQ ID NO: 83) byHindIII restriction enzyme digest and inserted into the F14.5L transfervector, pNCVVf14.51T (SEQ ID NO: 11), precut with HindIII to generateplasmid FSEL-IL24-1 (SEQ ID NO: 86). The SEL-IL-24 expression cassettewas confirmed by sequencing.

m. FSL-IL-24: For Insertion of an Expression Cassette Encoding IL-24Under the Control of the Vaccinia P_(SL) Promoter into the VacciniaF14.5L Locus.

Vector FSL-IL24-1 (SEQ ID NO: 85) was employed to develop strainGLV-1h98 having the following genotype: F14.5L: (P_(SL))IL-24, TK:(P_(SEL))rTrfR-(P_(7.5k))LacZ, HA: (P_(11k))gusA. Strain GLV-1h98 wasgenerated by inserting DNA encoding the human IL-24 protein operablylinked to the vaccinia virus synthetic late promoter (P_(SL)) (SEQ IDNO: 38) into the F14.5L locus of starting strain GLV-1h68, therebydeleting the Ruc-GFP fusion gene expression cassette at the F14.5L locusof GLV-1 h68. Vector FSL-IL24-1 contains a DNA fragment encoding theIL-24 protein operably linked to the vaccinia synthetic late promoter(P_(SL)) and sequences of the F14.5L gene flanking the (P_(SL))-fusionprotein-encoding DNA fragment.

Plasmid pCR-SL3 (SEQ ID NO: 39; see (j) above for description ofpCR-SL3), containing the vaccinia synthetic late promoter, i.e., P_(SL),was used as the source of the vaccinia synthetic late promoter ingenerating vector FSL-IL24-1.

To generate vector FSL-IL24-1, the IL-24 cDNA molecule (SEQ ID NO: 107)was released from vector pCRII-IL24-3 (see (n) above; SEQ ID NO: 80) byKpnI and SalI restriction enzyme digest and inserted into vector pCR-SL3(SEQ ID NO: 39), precut with SalI and SmaI to generate plasmidpCR-SL-IL24-2 (SEQ ID NO: 82), thus placing the IL-24 fusion cDNA underthe control of vaccinia virus synthetic late (SL) promoter.

The cDNA of SL-IL-24 was released from pCR-SL-IL24-2 (SEQ ID NO: 82) byHindIII and BamHI restriction enzyme digest and inserted into the F14.5Ltransfer vector, pNCVVf14.51T (SEQ ID NO: 11), precut with HindIII andBamHI to generate plasmid FSL-IL24-1 (SEQ ID NO: 85). The SL-IL-24expression cassette was confirmed by sequencing.

n. pCR-TK-SE-tTF-RGD: for Insertion of an Expression Cassette Encodingthe tTF-RGD Fusion Protein Under the Control of the Vaccinia P_(SE)Promoter into the Vaccinia TK Locus

Vector pCR-TK-SE-tTF-RGD (SEQ ID NO: 95) was employed to develop strainGLV-1h104 having the following genotype: F14.5L: (P_(SEL))Ruc-GFP; TK:(P_(SE))tTF-RGD; HA: (P_(11k))gusA. Strain GLV-1h104 was generated byinserting DNA encoding a tTF-RGD fusion protein (SEQ ID NO: 92 (DNAsequence); SEQ ID NO: 93 (amino acid sequence)) into the TK locus ofstrain GLV-1h68 thereby deleting the rTrfR-LacZ expression cassette atthe TK locus of strain GLV-1h68. Vector pCR-TK-SE-tTF-RGD contains a DNAfragment encoding the tTF-RGD fusion protein operably linked to thevaccinia synthetic early promoter (P_(SE)), sequences of the TK geneflanking the (P_(SE))-fusion protein-encoding DNA fragment, the E. coliguanine phosphoribosyltransferase (gpt) gene under the control of thevaccinia virus P7.5 k early and late promoter for transient dominantselection of virus that has incorporated the vector, and sequences ofthe pUC plasmid.

cDNA encoding human tissue factor (huTF) was synthesized from RNAextracted from MCF-7 cells (Qiagen RNA extraction kit). The huTF cDNAwas synthesized from the RNA in a reverse transcriptase reaction(Invitrogen Superscript II cDNA synthesis kit) using primerhu-tTF-RGD-rev-cDNA, which binds to a region upstream of the huTFsequence:

hu-tTF-RGD-rev-cDNA 5′-CTTTCTACACTTGTGTAGAGATATAGC-3′ (SEQ ID NO: 91)

After cDNA synthesis, the tTF-RGD fragment was PCR amplified (InvitrogenAccu Prime Pfx Supermix) using hu-TF cDNA as a template and thefollowing primers:

hu-tTF-RGD-for (SalI) (SEQ ID NO: 115)5′-GTCGACCCACCATGGAGACCCCTGCCTG-3′ and hu-tTF-RGD-rev (PacI) (SEQ ID NO:116) 5′-TTAATTAATATTATGGAGAATCACCTCTTCCTCTGAATTCCCCTT TCTCCTGG-3′.The hu-tTF-RGD-rev primer contains additional restriction endonucleasesites and the sequence of the RGD binding motif.

The PCR product was cloned into vector pCR-Blunt II-TOPO (Invitrogen;SEQ ID NO: 40) via blunt end ligation (Quick Ligation Kit; New EnglandBiolabs) to yield pCRII-tTF-RGD (SEQ ID NO: 94). The tTF-RGD cDNAmolecule (SEQ ID NO: 92) was confirmed by sequencing.

The vaccinia synthetic early promoter, i.e., P_(SE), and flanking TKgene regions of pCR-TK-SE-tTF-RGD are derived from an intermediateplasmid, TK-SE-CSF-2 (SEQ ID NO: 110), which contains the cDNA forGM-CSF under the control of the vaccinia synthetic early promoterflanked by the TK gene regions. pCR-SE1 (SEQ ID NO: 36; see (h) abovefor description of pCR-SE1), containing the vaccinia synthetic earlypromoter, i.e., P_(SE), was used as the source of the vaccinia syntheticearly promoter in generating vector TK-SE-CSF-2. The cDNA encodingGM-CSF protein (mouse granulocyte-macrophage colony-stimulating factor)was PCR amplified from pPICZA-mGM-CSF (SEQ ID NO: 72) with the primersGM-CSF5 5′-CTAGTCGACATGTGGCTGCAGAATTTACTTTTCCTGGGCATTGTGGTCTACAGCCTCTCAGCACCCACCCGCTCACCCATC-3′ (SEQ ID NO: 70), containing thesignal peptide sequence, and

GM-CSF3

5′-GGGTCATTTTTGGACTGGTTTTT-3′ (SEQ ID NO: 71), containing a stop codon.The PCR amplification product was cloned into vector pCR-Blunt 1′-TOPO(SEQ ID NO: 40; Invitrogen, Carlsbad, Calif.). The resulting vectorpCRII-CSF9 (SEQ ID NO: 108), which contained the correct insert, wasdigested with SalI and EcoRI (blunt-ended after digestion), and thereleased GM-CSF cDNA was cloned into vector pCR-SE1 (SEQ ID NO: 36)precut with SalI and SmaI, resulting in SE-CSF-2 (SEQ ID NO: 109). Thus,SE-CSF-2 contains the vaccinia synthetic early promoter (P_(SE))operably linked to DNA encoding GM-CSF. The GM-CSF expression cassettecontaining GM-CSF cDNA under the control of the P_(SE) was excised fromSE-CSF-2 by SacI digestion and cloned into the same-cut vectorpCR-TKLR-gpt2 (SEQ ID NO: 17) to generate the construct TK-SE-CSF-2 (SEQID NO: 110). This cloning step places the (P_(SE))GM-CSF expressioncassette between the left and right TK gene flanking sequences inpCR-TKLR-gpt2 and eliminates the non-coding DNA that is located betweenthese flanking sequences in pCR-TKLR-gpt2.

To generate vector pCR-TK-SE-tTF-RGD, the tTF-RGD fragment was releasedby SalI and PacI restriction enzyme digest of pCRII-tTF-RGD (SEQ ID NO:94) and inserted into TK-SE-CSF-2 (SEQ ID NO: 110), precut with SalI andPacI, to generate plasmid pCR-TK-SE-tTF-RGD (SEQ ID NO: 95), thusplacing the tTF-RGD cDNA under the control of vaccinia virus syntheticearly (P_(SE)) promoter and in between the left and right TK geneflanking sequences. The tTF-RGD cDNA insert was confirmed by sequencing.

o. pCR-TK-SEL-tTF-RGD: for Insertion of an Expression Cassette Encodingthe tTF-RGD Fusion Protein Under the Control of the Vaccinia P_(SEL)Promoter into the Vaccinia TK Locus

Vector pCR-TK-SEL-tTF-RGD (SEQ ID NO: 96) was employed to develop strainGLV-1h105 having the following genotype: F14.5L: (P_(SEL))Ruc-GFP; TK:(P_(SEL))tTF-RGD; HA: (P_(11k))gusA. Strain GLV-1h105 was generated byinserting DNA encoding a tTF-RGD fusion protein (SEQ ID NO: 92) into theTK locus of strain GLV-1h68 thereby deleting the rTrfR-LacZ expressioncassette at the TK locus of strain GLV-1h68. Vector pCR-TK-SEL-tTF-RGDcontains a DNA fragment encoding the tTF-RGD fusion protein operablylinked to the vaccinia synthetic early/late promoter (P_(SEL)),sequences of the TK gene flanking the (P_(SEL))-fusion protein-encodingDNA fragment, the E. coli guanine phosphoribosyltransferase (gpt) geneunder the control of the vaccinia virus P7.5 k early and late promoterfor transient dominant selection of virus that has incorporated thevector and sequences of the pUC plasmid.

The vaccinia synthetic early/late promoter, i.e., P_(SEL), and flankingTK gene regions of pCR-TK-SEL-tTF-RGD are derived from an intermediateplasmid, TK-SEL-CSF-2 (SEQ ID NO: 112), which contains the cDNA forGM-CSF under the control of the vaccinia synthetic early/late promoterflanked by the TK gene regions. Plasmid pCR-SEL4 (SEQ ID NO: 33; see (f)above for construction of pCR-SEL4), containing the vaccinia syntheticearly/late promoter, i.e., P_(SEL), was used as the source of thevaccinia synthetic early/late in generating vector TK-SEL-CSF-2. DNAencoding GM-CSF was excised from pCRII-CSF9 (SEQ ID NO: 108) with SalIand EcoRI (blunt-ended after digestion), and cloned into vector pCR-SEL4(SEQ ID NO: 33) precut with SalI and SmaI, resulting in SEL-CSF-2 (SEQID NO: 111). Thus, SEL-CSF-2 contains the vaccinia synthetic early/latepromoter (P_(SEL)) operably linked to DNA encoding GM-CSF. The GM-CSFexpression cassette containing DNA encoding GM-CSF under the control ofP_(SEL) was then excised from SEL-CSF-2 by SacI digestion and clonedinto the same-cut vector pCR-TKLR-gpt2 (SEQ ID NO: 17) to generate theconstruct TK-SEL-CSF-2 (SEQ ID NO: 112). This cloning step places the(P_(SEL))GM-CSF expression cassette between the left and right TK geneflanking sequences in pCR-TKLR-gpt2 and eliminates the non-coding DNAthat is located between these flanking sequences in pCR-TKLR-gpt2.

To generate vector pCR-TK-SEL-tTF-RGD, the tTF-RGD fragment was releasedby SalI and PacI restriction enzyme digest of pCRII-tTF-RGD (see (n)above; SEQ ID NO: 94) and inserted into TK-SEL-CSF-2 (SEQ ID NO: 112),precut with SalI and PacI to generate plasmid pCR-TK-SEL-tTF-RGD (SEQ IDNO: 96), thus placing the tTF-RGD cDNA under the control of vacciniavirus synthetic early/late (P_(SEL)) promoter and in between the leftand right TK gene flanking sequences. The tTF-RGD cDNA insert wasconfirmed by sequencing.

p. pCR-TK-SL-tTF-RGD: for Insertion of an Expression Cassette Encodingthe tTF-RGD Fusion Protein Under the Control of the Vaccinia P_(SL)Promoter into the Vaccinia TK Locus

Vector pCR-TK-SL-tTF-RGD (SEQ ID NO: 97) was employed to develop strainGLV-1h106 having the following genotype: F14.5L: (P_(SEL))Ruc-GFP; TK:(P_(SL))tTF-RGD; HA: (P_(11k))gusA. Strain GLV-1h106 was generated byinserting DNA encoding a tTF-RGD fusion protein (SEQ ID NO: 92) into theTK locus of strain GLV-1h68 thereby deleting the rTrfR-LacZ expressioncassette at the TK locus of strain GLV-1h68. Vector pCR-TK-SL-tTF-RGDcontains a DNA fragment encoding the tTF-RGD fusion protein operablylinked to the vaccinia synthetic late promoter (P_(SL)), sequences ofthe TK gene flanking the (P_(SL))-fusion protein-encoding DNA fragment,the E. coli guanine phosphoribosyltransferase (gpt) gene under thecontrol of the vaccinia virus P7.5 k early and late promoter fortransient dominant selection of virus that has incorporated the vector,and sequences of the pUC plasmid.

The vaccinia synthetic late promoter, i.e., P_(SL), and flanking TK generegions of pCR-TK-SL-tTF-RGD are derived from an intermediate plasmid,TK-SL-CSF-2 (SEQ ID NO: 114), which contains the cDNA for GM-CSF underthe control of the vaccinia synthetic late promoter flanked by the TKgene regions.

Plasmid pCR-SL3 (SEQ ID NO: 39; see (j) above for description ofpCR-SL3), containing the vaccinia synthetic late promoter, i.e., P_(SL),was used as the source of the vaccinia synthetic late promoter ingenerating vector TK-SL-CSF-3 (SEQ ID NO: 114). DNA encoding mouseGM-CSF was excised from pCRII-CSF9 (SEQ ID NO: 108) with SalI and EcoRI(blunt-ended after digestion), and cloned into vector pCR-SL3 (SEQ IDNO: 39) precut with SalI and SmaI, resulting in SL-CSF-2 (SEQ ID NO:113). Thus, SL-CSF-2 contains the vaccinia synthetic late promoter(P_(SL)) operably linked to DNA encoding GM-CSF. The GM-CSF expressioncassette containing DNA encoding GM-CSF under the control of the P_(SL)was excised out from SL-CSF-2 by Sac I and cloned into the same-cutvector pCR-TKLR-gpt2 (SEQ ID NO: 17) to generate the constructTK-SL-CSF-3 (SEQ ID NO: 114). This cloning step places the(P_(SL))GM-CSF expression cassette between the left and right TK geneflanking sequences in pCR-TKLR-gpt2 and eliminates the non-coding DNAthat is located between these flanking sequences in pCR-TKLR-gpt2.

To generate vector pCR-TK-SL-tTF-RGD, the tTF-RGD fragment was releasedby SalI and PacI restriction enzyme digest of pCRII-tTF-RGD (see (n)above; SEQ ID NO: 94) and inserted into TK-SL-CSF-3 (SEQ ID NO: 114),precut with SalI and PacI to generate plasmid pCR-TK-SL-tTF-RGD (SEQ IDNO: 97), thus placing the tTF-RGD cDNA under the control of vacciniavirus synthetic late (P_(SL)) promoter and in between the left and rightTK gene flanking sequences. The tTF-RGD cDNA insert was confirmed bysequencing.

q. pCR-TK-SE-G6-FLAG: for Insertion of an Expression Cassette Encodingthe G6-FLAG Fusion Protein Under the Control of the Vaccinia P_(SE)Promoter into the Vaccinia TK Locus

Vector pCR-TK-SE-G6-FLAG (SEQ ID NO: 100) was employed to develop strainGLV-1h107 having the following genotype: F14.5L: (P_(SEL))Ruc-GFP; TK:(P_(SE)) G6-FLAG; HA: (P_(11k))gusA. Strain GLV-1h107 was generated byinserting DNA encoding a G6-FLAG fusion protein (SEQ ID NO: 99; G6 isthe anti-VEGF scAb) into the TK locus of strain GLV-1h68 therebydeleting the rTrfR-LacZ expression cassette at the TK locus of strainGLV-1h68. Vector pCR-TK-SE-G6-FLAG contains a DNA fragment encoding theG6-FLAG fusion protein operably linked to the vaccinia synthetic earlypromoter (P_(SE)), sequences of the TK gene flanking the (P_(SE))-fusionprotein-encoding DNA fragment, the E. coli guaninephosphoribosyltransferase (gpt) gene under the control of the vacciniavirus P7.5 k early and late promoter for transient dominant selection ofvirus that has incorporated the vector, and sequences of the pUCplasmid.

cDNA encoding G6-FLAG was obtained from vector pGA4-G6 (GeneArt; SEQ IDNO: 98). The vector contains DNA encoding an artificially synthesizedsingle chain antibody (scAb) directed against VEGF (scFv anti-VEGF). Thegene encodes the kappa light chain leader sequence for the secretion ofthe protein, the sequence of the V_(H) domain of the scAb followed by alinker sequence and the sequence of the V_(L) domain of the scAb. TheC-terminal end of the gene is fused to DNA encoding a FLAG-tag for easeof protein detection. The 5′ end the G6-FLAG fragment contains a SalIsite, and the 3′ end contains a PacI site.

To generate vector pCR-TK-SE-G6-FLAG, the G6-FLAG fragment was releasedby SalI and PacI restriction enzyme digest of pGA4-G6 (SEQ ID NO: 98)and inserted into TK-SE-CSF-2 (see (n) above; SEQ ID NO: 110), precutwith SalI and PacI, to generate plasmid pCR-TK-SE-G6-FLAG (SEQ ID NO:100), thus placing the G6-FLAG cDNA under the control of vaccinia virussynthetic early (P_(SE)) promoter and in between the left and right TKgene flanking sequences. The G6-FLAG cDNA insert was confirmed bysequencing.

r. pCR-TK-SEL-G6-FLAG: for Insertion of an Expression Cassette Encodingthe G6-FLAG Fusion Protein Under the Control of the Vaccinia P_(SEL)Promoter into the Vaccinia TK Locus

Vector pCR-TK-SEL-G6-FLAG (SEQ ID NO: 101) was employed to developstrain GLV-1h108 having the following genotype: F14.5L:(P_(SEL))RUC-GFP; TK: (P_(SEL))G6-FLAG; HA: (P_(11k))gusA. StrainGLV-1h108 was generated by inserting DNA encoding a G6-FLAG fusionprotein (SEQ ID NO: 99) into the TK locus of strain GLV-1h68 therebydeleting the rTrfR-LacZ expression cassette at the TK locus of strainGLV-1h68. Vector pCR-TK-SEL-G6-FLAG contains a DNA fragment encoding theG6-FLAG fusion protein operably linked to the vaccinia syntheticearly/late promoter (P_(SEL)), sequences of the TK gene flanking the(P_(SEL))-fusion protein-encoding DNA fragment, the E. coli guaninephosphoribosyltransferase (gpt) gene under the control of the vacciniavirus P7.5 k early and late promoter for transient dominant selection ofvirus that has incorporated the vector, and sequences of the pUCplasmid.

To generate vector pCR-TK-SEL-G6-FLAG, the G6-FLAG fragment was releasedby SalI and PacI restriction enzyme digest of pGA4-G6 (see (t) above;SEQ ID NO: 98) and inserted into TK-SEL-CSF-2 (see (o) above; SEQ ID NO:112), precut with SalI and PacI, to generate plasmid pCR-TK-SEL-G6-FLAG(SEQ ID NO: 101), thus placing the tTF-RGD cDNA under the control ofvaccinia virus synthetic early/late (P_(SEL)) promoter and in betweenthe left and right TK gene flanking sequences. The G6-FLAG cDNA insertwas confirmed by sequencing.

s. pCR-TK-SL-G6-FLAG: for Insertion of an Expression Cassette Encodingthe G6-FLAG Fusion Protein Under the Control of the Vaccinia P_(SL)Promoter into the Vaccinia TK Locus

Vector pCR-TK-SL-G6-FLAG (SEQ ID NO: 102) was employed to develop strainGLV-1h109 having the following genotype: F14.5L: (P_(SEL))Ruc-GFP; TK:(P_(SL)) G6-FLAG; HA: (P_(11k))gusA. Strain GLV-1h109 was generated byinserting DNA encoding a G6-FLAG fusion protein (SEQ ID NO: 99) into theTK locus of strain GLV-1h68 thereby deleting the rTrfR-LacZ expressioncassette at the TK locus of strain GLV-1h68. Vector pCR-TK-SL-G6-FLAGcontains a DNA fragment encoding the G6-FLAG fusion protein operablylinked to the vaccinia synthetic late promoter (P_(SL)), sequences ofthe TK gene flanking the (P_(SL))-fusion protein-encoding DNA fragment,the E. coli guanine phosphoribosyltransferase (gpt) gene under thecontrol of the vaccinia virus P7.5 k early and late promoter fortransient dominant selection of virus that has incorporated the vector,and sequences of the pUC plasmid.

To generate vector pCR-TK-SL-G6-FLAG, the G6-FLAG fragment was releasedby SalI and PacI restriction enzyme digest of pGA4-G6 (see (t) above;SEQ ID NO: 98) and inserted into TK-SL-CSF-3 (see (p) above; SEQ ID NO:114), precut with SalI and PacI to generate plasmid pCR-TK-SL-G6-FLAG(SEQ ID NO: 102), thus placing the G6-FLAG cDNA under the control ofvaccinia virus synthetic late (P_(SL)) promoter and in between the leftand right TK gene flanking sequences. The G6-FLAG cDNA insert wasconfirmed by sequencing.

t. pF14.5-SEL-RG: for Insertion of an Expression Cassette Encoding theRuc-GFP Fusion Protein Under the Control of the Vaccinia P_(SEL)Promoter into the Vaccinia F14.5L Locus

pF14.5-SEL-RG (SEQ ID NO: 104) is a targeting vector that can beemployed to facilitate insertion of foreign genes in the F14.5L locus ofLIVP.

The ruc-gfp fusion cDNA from pcDNA-RG (see, for example, Wang et al.,2002) was amplified by PCR using AccuPrime pfx SuperMix (Invitrogen),using primer that comprise the vaccinia synthetic early/late promoter(P_(SEL)), which places the ruc-gfp under the control of P_(SEL)promoter:

(SEQ ID NO: 117) 5′-ATCAAGCTTAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATGACTTCGAAAGTTTATGATCCAGAAC-3′ and (SEQ ID NO: 118)5′-TCACTTGTACAGCTCGTCCA-3′.

The resulting PCR product was cloned into pCR-Blunt 1′-TOPO vector(Invitrogen; SEQ ID NO: 40) to yield pCRII-SEL-RG (SEQ ID NO: 105). Thevector was sequence confirmed.

To generate vector pF14.5-SEL-RG, the SEL-RG cDNA fragment was releasedfrom pCRII-SEL-RG (SEQ ID NO: 105) by Hind III and EcoR V restrictionenzyme digest and inserted into pNCVVf14.51T (SEQ ID NO: 11), precutwith HindIII and BamHI (blunt ended) to generate plasmid pF14.5-SEL-RG(SEQ ID NO: 104), thus placing the Ruc-GFP fusion cDNA under the controlof vaccinia virus synthetic early/late (P_(SEL)) promoter and in betweenthe left and right F14.5L gene flanking sequences.

3. Preparation of Recombinant Vaccinia Viruses

a. GLV-1i69

CV-1 (African green monkey kidney fibroblast) cells (ATCC No. CCL-70),grown in DMEM (Mediatech, Inc., Herndon, Va.) with 10% FBS, wereinfected with GLV-1h68 at multiplicity of infection (m.o.i.) of 0.1 for1 hour, then transfected using Lipofectamine 2000 (Invitrogen, Carlsbad,Calif.) with PCR-amplified A34R (SEQ ID NO: 58) coding sequence from VVIHD-J using the following primers: 5′-CATTAATAAATGAAATCGCTTAATAG-3′ (SEQID NO: 59) and 5′-GGCGGCGTACGTTAACGAC-3′ (SEQ ID NO: 60). Recombinantvirus was selected based on its comet-like plaque morphology asdescribed below.

Two days after transfection, the medium was harvested. To enrich therecombinant extracellular enveloped viruses (EEVs) (i.e. to increase thepercentage of recombinant EEV within the infected medium), CV-1 cellswere infected with the infected/transfected medium. Two days postinfection the infected medium was collected. After the fourth round ofthe enrichment, the infected medium was diluted and used to infect CV-1cells. Ten well-isolated plaques were picked and purified for a total ofthree times. Eight of ten isolates formed comet-like plaques underliquid medium.

b. GLV-1h and GLV-1j Series

CV-1 cells, grown in DMEM (Mediatech, Inc., Hemdon, Va.) with 10% FBS,were infected with the indicated parental viruses (Table 2) at m.o.i. of0.1 for 1 hr, then transfected using Lipofectamine 2000 or Fugene(Roche, Indianapolis, Ind.) with 2 μg of the corresponding transfervector (Table 2). Infected/transfected cells were harvested and therecombinant viruses were selected using a transient dominant selectionsystem and plaque purified using methods known in the art (see, e.g.,Falkner and Moss, J. Virol., 64, 3108-3111 (1990)). Isolates were plaquepurified five times with the first two rounds of plaque isolationconducted in the presence of mycophenolic acid, xanthine andhypoxanthine which permits growth only of recombinant virus thatexpressing the selectable marker protein, i.e., E. coli guaninephosphoribosyltransferase (gpt), under the control of the vacciniaP_(7.5kE) promoter. As described herein, each of the transfer vectorsused in the generation of the GLV-1h and GLV-1j series of recombinantvaccinia virus contained a (P_(7.5kE))gpt expression cassette. Thus,growth of the virus in the presence of the selection agents enabledidentification of virus in which the first crossover event of homologousrecombination between the transfer vector and the parental strain genomehad occurred. Subsequent growth of the isolates in the absence ofselection agents and further plaque purification yielded isolates thathad undergone a second crossover event resulting in deletion of the DNAencoding guanine phosphoribosyltransferase from the genome. This wasconfirmed by the inability of these isolates to grow in the presence ofselection agents.

4. Verification of Vaccinia Virus Strain Genotypes

The genotypes of the modified vaccinia virus strains were verified byPCR and restriction enzyme digestion. The nucleotide sequence of thecoding sequence from the IHD-J A34R gene (SEQ ID NO: 58) in GLV-1i69 wasfurther verified by sequencing. Lack of expression of the gusA gene inGLV-1h70, GLV-1h73, GLV-1 h74, GLV-1h82, GLV-1h83, GLV-1h84, GLV-1h86,GLV-1h90, GLV-1h91 and GLV-1h92 was confirmed by X-GlcA(5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid) staining of theinfected cells. Viruses lacking gusA expression are unable to convertthe X-GlcA substrate as indicated by lack of development of blue colorin the assay as compared to a control strain (e.g. GLV-1h68). Lack ofexpression of the GFP gene in GLV-1h71, GLV-1h73, GLV-1h74, GLV-1h84,GLV-1h85, GLV-1h96, GLV-1h97 and GLV-1h98 was confirmed by fluorescencemicroscopy as compared to a control strain (e.g. GLV-1h68). Lack ofexpression of β-galactosidase in GLV-1 h72, GLV-1h74, GLV-1h81,GLV-1h84, GLV-1h85, GLV-1h86, GLV-1h104, GLV-1h105, GLV-1h106,GLV-1h107, GLV-1h108 and GLV-1h109 was confirmed by X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) staining of theinfected cells. Viruses lacking lacZ expression are unable to convertthe X-gal substrate as indicated by lack of development of blue color inthe assay as compared to a control strain (e.g. GLV-1h68). Standardtechniques for X-GlcA and X-gal viral staining and fluorescencemicroscopy were employed and are well-known in the art.

Expression of mRFP in GLV-1h84 was confirmed using a Leica DMI 6000 Bfluorescence microscope at 2 days post-infection of CV-1 cells andcompared to mock infected cells or non-mRFP expression strains (e.g.,GLV-1h73). In one example, the GLV-1h84 infected cells expressed over2.2×10¹⁰ relative light units compared to no expression in the GLV-1h73strain. Expression of firefly luciferase in GLV-1h84 was confirmed attwo days post-infection of CV-1 cells performed using the Chroma-Gloluciferase assay systems (Promega) and relative light units (RLU) weremeasured using a Turner TD-20e luminometer.

B. Vaccinia Virus Purification

Ten T225 flasks of confluent CV-1 cells (seeded at 2×10⁷ cells per flaskthe day before infection) were infected with each virus at m.o.i. of0.1. The infected cells were harvested two days post infection and lysedusing a glass Dounce homogenizer. The cell lysate was clarified bycentrifugation at 1,800 g for 5 min, and then layered on a cushion of36% sucrose, and centrifuged at 13,000 rpm in a HB-6 rotor, SorvallRC-5B Refrigerated Superspeed Centrifuge for 2 hours. The virus pelletwas resuspended in 1 ml of 1 mM Tris, pH 9.0, loaded on a sterile 24% to40% continuous sucrose gradient, and centrifuged at 26,000 g for 50 min.The virus band was collected and diluted using 2 volumes of 1 mM Tris,pH 9.0, and then centrifuged at 13,000 rpm in a HB-6 rotor for 60 min.The final virus pellet was resuspended in I ml of 1 mM Tris, pH 9.0 andthe titer was determined in CV-1 cells (ATCC No. CCL-70).

Example 2 In Vitro Virus Infection Studies A. Cell Lines Employed forVirus Infection

A549 (human lung carcinoma, ATCC No. CCL-185), CV-1 (African greenmonkey kidney fibroblast, ATCC No. CCL-70), MRC-5 (human lungfibroblast, ATCC No. CCL-171), Vero (African green monkey kidneyepithelial, ATCC No. CCL-81) and 293 (human kidney fibroblast, ATCC No.CRL-1573) cells were obtained from the American Type Culture Collection(ATCC; Manassas, Va.). G1-101A (human breast tumor) cells (obtained fromDr. Alex Aller, Rumbaugh—Goodwin Institute for Cancer Research, Inc.,Plantation, Fla.) were derived from G101, a human ductal adenocarcinomacell line (Rathinavelu et al., Cancer Biochem. Biophys., 17:133-146(1999)). Primary chick embryo fibroblasts (CEF) were prepared from10-day-old embryos and grown in Ham's F-10 (Biowhittaker, Walkersville,Md.)/199 (1:1, Mediatech, Inc., Herndon, Va.) supplemented with 5%heat-inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, Calif.),2% nonessential amino acids (NEAA, Mediatech, Inc., Herndon, Va.) and 1%antibiotic-antimycotic solution (Mediatech, Inc., Herndon, Va.). A549cells were cultured in RPMI-1640 (Mediatech, Inc., Herndon, Va.)supplemented with 10% FBS. CV-1 cells were grown in DMEM (Mediatech,Inc., Herndon, Va.) with 10% FBS. MRC-5, Vero, and 293 cells werecultured in EMEM (Mediatech, Inc., Herndon, Va.) supplemented with 10%FBS, 1% NEAA and 1% sodium pyruvate (Sigma, St. Louis, Mo.). GI-01Acells were grown in RPMI 1640 with 20% FBS, 1% antibiotic-antimycoticsolution, 10 mM HEPES, 1% sodium pyruvate, 5 ng/ml of β-estradiol(Sigma, St. Louis, Mo.), and 5 ng/ml of progesterone (Sigma, St. Louis,Mo.). All cell lines were maintained at 37° C. with 5% CO₂ in ahumidified incubator.

B. Analysis of Viral Yields in CV-1 Cells

The ability of modified vaccinia virus strains to infect and replicatein vitro was analyzed by measuring plaque forming units (PFU) producedfollowing infection of CV-1 cells with purified recombinant virus, atechnique well-known in the art. 2×10⁸ CV-1 cells were infected witheach virus at m.o.i. of 0.1 for 1 hour at 37° C. and harvested 2 dayspost infection. Each virus was purified through sucrose gradient andsubjected to a plaque forming assay using CV-1 cells. Yields of purifiedvirus for exemplary modified vaccinia virus strains are shown in Table3.

TABLE 3 Purified Virus Virus Yield^(a) GLV-1h68 1.1 × 10⁹ GLV-1h70 1.1 ×10⁹ GLV-1h71 2.1 × 10⁹ GLV-1h72 1.9 × 10⁹ GLV-1h73 4.1 × 10⁹ GLV-h74 4.6× 10⁹ ^(a)Yield in PFU/2 × 10⁸ cells at 2 days post infection

C. Virus Production in Different Cell Lines Experiment 1 Comparison ofVirus Production in Different Cell Lines

A549, CEF, CV-1, MRC-5, Vero and 293 cells in 6-well plates wereinfected with GLV-1h68 or GLV-1h74 at m.o.i. of 0.01 for 1 hour at 37°C. The inoculum was aspirated and the cell monolayers were washed twicewith 2 ml of DPBS (Mediatech, Inc., Herndon, Va.). Two ml of cellculture medium were added into each well. Three wells of each cell typewere harvested at 24 h, 48 h and 72 h post infection (PI), respectively.The virus titer in crude cell lysates from infected cells was determinedin CV-1 cells. Data for yields of exemplary viruses GLV-1h68 andGLV-1h74 in different cell lines are shown in Table 4 and Table 5,respectively.

GLV-1h68 yields were high in all cells tested except for CEF cells. Thevirus yields in CV-1 and A549 cells on day 3 post-infection were quitesimilar, but slightly higher than that in MRC-5 cells, more than 3 timesas high as that in Vero and 293 cells, and more than 1800 times as highas that in CEF cells. The cell lines that provided for significant virusyields are potential candidate cell lines for the GMP production ofGLV-1h68. Since vaccinia virus has a very broad host range in vitrothere can be other cell lines that support GLV-1h68 replication as wellas, or better than, the cell lines tested, which can be used for the GMPproduction of GLV-1h68.

GLV-1h74 yields were high in all cells tested, including CEF cells. Thehighest yields of virus were obtained with CV-1 cells and A549 cells.The virus yield in CV-1 cells on day 3 post-infection was more than 1.7times as high as that in A549 cells, about 3.5 times as high as that inMRC-5 and Vero cells, 6.2 times as high as that in 293 cells, and about52 times as high as that in CEF cells. The yields of GLV-1h74 in all sixcell lines tested were higher than the yields of GLV-1h68. The virusyields of GLV-1h74 in A549, CV-1, MRC-5, Vero and 293 cells were 3 to 8times as high as that of GLV-1h68 in the same cell lines. Strikingly,the virus yield of GLV-1h74 in CEF cells was 278 times higher than thatof GLV-1h68 in the same cell line. All cell lines tested are potentialcandidate cell lines for the GMP production of GLV-1h74 since they allsupported GLV-1h74 replication very well. Since vaccinia virus has avery broad host range in vitro there can be other cell lines that cansupport GLV-1h74 replication as well as the cell lines tested or everbetter, which can be used for the GMP production of GLV-1h74.

TABLE 4 Virus yields of GLV-1h68 in different cell lines Virus Yield(PFU/10⁶ Cells) Cell Type Day 0 Day 1 Day 2 Day 3 A549 10⁴ ± 0 4.3 × 10⁶± 5.3 × 10⁴ 6.1 × 10⁷ ± 7.6 × 10⁶ 8.1 × 10⁷ ± 1.1 × 10⁷ CEF 10⁴ ± 0 1.8× 10³ ± 4.6 × 10² 3.4 × 10⁴ ± 6.3 × 10³ 4.3 × 10^(4 ±) 1.5 × 10⁴ CV 110⁴ ± 0 6.3 × 10⁵ ± 2.4 × 10⁴ 5.7 × 10⁷ ± 8.9 × 10⁶ 1.0 × 10⁸ ± 2.1 ×10⁶ MRC-5 10⁴ ± 0 2.9 × 10⁵ ± 4.0 × 10⁴ 3.7 × 10⁷ ± 5.0 × 10⁶ 6.2 × 10⁷± 5.8 × 10⁵ Vero 10⁴ ± 0 5.7 × 10⁴ ± 8.8 × 10³ 1.2 × 10⁶ ± 1.8 × 10⁵ 2.4× 10⁷ ± 4.0 × 10⁶ 293 10⁴ ± 0 2.0 × 10⁵ ± 6.4 × 10⁴ 1.5 × 10⁷ ± 4.2 ×10⁶ 2.8 × 10⁷ ± 7.3 × 10⁶

TABLE 5 Virus yields of GLV-1h74 in different cell lines Virus Yield(PFU/10⁶ Cells) Cell Type Day 0 Day 1 Day 2 Day 3 A549 10⁴ ± 0 3.0 × 10⁷± 2.2 × 10⁶ 2.6 × 10⁸ ± 4.7 × 10⁷ 3.6 × 10⁸ ± 3.8 × 10⁷ CEF 10⁴ ± 0 3.2× 10⁴ ± 7.6 × 10³ 5.9 × 10⁶ ± 8.0 × 10⁵ 1.2 × 10⁷ ± 3.6 × 10⁶ CV 1 10⁴ ±0 6.5 × 10⁶ ± 7.0 × 10⁵ 3.2 × 10⁸ ± 6.3 × 10⁷ 6.2 × 10⁸ ± 3.5 × 10⁷MRC-5 10⁴ ± 0 3.1 × 10⁶ ± 1.2 × 10⁵ 1.5 × 10⁸ ± 9.7 × 10⁶ 1.7 × 10⁸ ±3.8 × 10⁷ Vero 10⁴ ± 0 2.8 × 10⁶ ± 2.3 × 10⁵ 8.3 × 10⁷ ± 7.0 × 10⁶ 1.8 ×10⁸ ± 9.4 × 10⁶ 293 10⁴ ± 0 2.9 × 10⁷ ± 5.6 × 10⁶ 8.6 × 10⁷ ± 1.1 × 10⁷1.0 × 10⁸ ± 1.1 × 10⁷

Experiment 2 Comparison of Modified Vaccinia Strains

CEF, MRC-5, or GI-101A cells in 6-well plates were infected withGLV-1h68 or its derivatives at m.o.i. of 0.01 for 1 hour at 37° C. Theinoculum was aspirated and the cell monolayers were washed twice with 2ml of DPBS (Mediatech, Inc., Herndon, Va.). Two ml of cell culturemedium were added into each well. Three wells of each virus/cell typewere harvested at 24, 48, and 72 h post infection (PI), respectively.The crude cell lysates were titrated in CV-1 cells.

Based on the viral yields in CEF cells, the viruses tested can bedivided into two groups (Table 6). The virus yields in the first group(GLV-1h71, GLV-1h73, and GLV-1h74) were much better than those in thesecond group (GLV-1h68, GLV-1h70, and GLV-1h72). At all time points, theyields of the viruses in the first group were at least 10 times as highas that in the second group.

In the MRC-5 cell line, all viruses tested exhibited high virus yields(Table 7). In the first 24 hours, GLV-1h71, GLV-1h73, and GLV-1h74 hadhigher yields than did GLV-1h68, GLV-1h70 and GLV-1h72. On day 3, allviruses reached similar titers, except that the titer of GLV-1h74 wasabout 2 times as high as that of other viruses.

In the GI-101A cell line, all viruses except GLV-1h22 exhibited higheryields in the first day than did during the second day, and all virusesreached their maximum titers on day 2 (Table 8).

Overall, in all three cell lines tested, the virus lacking all foreigngene expression cassettes at all three loci (GLV-1h74) exhibited higheryields than did the virus lacking foreign gene expression cassettes attwo loci (GLV-1h73) and much better yields than did the viruses lackingforeign gene expression cassette(s) at only one locus (GLV-1h70, 71, and72). Also, all viruses had higher yields than their parental virus,GLV-1h68, indicating that foreign gene transcription and/or expressionreduced vaccinia virus growth in vitro. The more foreign gene expressioncassettes were replaced, the better the virus grew in vitro.Interestingly, among the viruses that have a foreign insert replaced atonly one locus, GLV-1h71 consistently had higher yields than GLV-1h72,whereas GLV-1h72 always showed higher yields than GLV-1h70. Ruc-GFPfusion gene expression cassette that was replaced in GLV-1h71 consistsof a synthetic early/later promoter that is stronger than the 11kpromoter directing GUS expression replaced in GLV-1h70. It appears thatstronger foreign gene expression exerts a stronger negative effect onvirus replication, although it cannot be ruled out that differentforeign proteins might have different effects on virus growth.Replacement of the insert at TK locus in GLV-1h72 that contains a strongsynthetic early/late promoter directing transcription of an anti-sensestrand of a transferrin receptor and a 7.5 k early/late promotercontrolling LacZ expression resulted in more enhanced virus replicationthan did replacing an insert containing 11k promoter directing GUSexpression in GLV-1h70, although the 11k promoter is much stronger than7.5 k promoter, indicating that transcription in the absence oftranslation also appeared to have negative effects on virus yields invitro.

TABLE 6 Virus Yields of Different Vaccinia Recombinants in CEF Cells PIVirus Yield (PFU/10⁶ Cells) (hr) GLV-1h68 GLV-1h70 GLV-1h71 GLV-1h72GLV-1h73 GLV-1h74 0 1.0 × 10⁴ 1.0 × 10⁴ 1.0 × 10⁴ 1.0 × 10⁴ 1.0 × 10⁴1.0 × 10⁴ 24 2.9 × 10³ ± 1.4 × 10³ ± 3.5 × 10⁴ ± 3.1 × 10³ ± 7.7 × 10⁴ ±1.1 × 10⁵ ± 9.1 × 10² 3.7 × 10² 5.9 × 10³ 6.0 × 10² 1.7 × 10⁴ 1.3 × 10⁴48 2.2 × 10⁵ ± 1.7 × 10⁵ ± 5.9 × 10⁶ ± 1.9 × 10⁵ ± 7.4 × 10⁶ ± 1.2 × 10⁷± 4.3 × 10⁴ 5.4 × 10⁴ 4.5 × 10⁵ 1.1 × 10⁵ 8.7 × 10⁵ 9.6 × 10⁶ 72 1.2 ×10⁶ ± 2.5 × 10⁶ ± 2.7 × 10⁷ ± 2.8 × 10⁶ ± 3.4 × 10⁷ ± 4.9 × 10⁷ ± 9.7 ×10⁴ 1.3 × 10⁶ 1.0 × 10⁷ 6.9 × 10⁵ 1.5 × 10⁷ 1.9 × 10⁷

TABLE 7 Virus Yields of Different Vaccinia Recombinants in MRC-5 CellsPI Virus Yield (PFU/10⁶ Cells) (hr) GLV-1h68 GLV-1h70 GLV-1h71 GLV-1h72GLV-1h73 GLV-1h74 0 1.0 × 10⁴ 1.0 × 10⁴ 1.0 × 10⁴ 1.0 × 10⁴ 1.0 × 10⁴1.0 × 10⁴ 24 9.9 × 10⁴ ± 9.9 × 10⁴ ± 6.0 × 10⁵ ± 1.2 × 10⁵ ± 1.2 × 10⁶ ±2.0 × 10⁶ ± 4.9 × 10³ 1.6 × 10⁴ 2.3 × 10⁵ 7.1 × 10³ 1.4 × 10⁵ 2.2 × 10⁵48 1.3 × 10⁷ ± 1.4 × 10⁷ ± 2.7 × 10⁷ ± 2.7 × 10⁷ ± 3.6 × 10⁷ ± 6.9 × 10⁷± 2.4 × 10⁶ 1.3 × 10⁶ 8.0 × 10⁵ 5.8 × 10⁶ 8.4 × 10⁶ 2.3 × 10⁷ 72 3.4 ×10⁷ ± 4.9 × 10⁷ ± 3.3 × 10⁷ ± 4.5 × 10⁷ ± 4.0 × 10⁷ ± 8.1 × 10⁷ ± 3.5 ×10⁶ 1.8 × 10⁷ 6.4 × 10⁶ 6.9 × 10⁶ 9.2 × 10⁶ 1.4 × 10⁷

TABLE 8 Virus Yields of Different Vaccinia Recombinants in GI-101A CellsPI Virus Yield (PFU/10⁶ Cells) (hr) GLV-1h22 GLV-1h68 GLV-1h70 GLV-1h71GLV-1h72 GLV-1h73 GLV-1h74 0 1.0 × 10⁴ 1.0 × 10⁴ 1.0 × 10⁴ 1.0 × 10⁴ 1.0× 10⁴ 1.0 × 10⁴ 1.0 × 10⁴ 24 2.2 × 10⁵ ± 3.0 × 10⁵ ± 3.5 × 10⁵ ± 1.1 ×10⁶ ± 3.9 × 10⁵ ± 2.5 × 10⁶ ± 23.7 × 10⁶ ± 3.3 × 10⁵  5.5 × 10³ 1.1 ×10⁴ 2.4 × 10⁴ 1.3 × 10⁵ 2.2 × 10⁴ 1.5 × 10⁵ 48 2.5 × 10⁶ ± 8.8 × 10⁵ ±1.5 × 10⁶ ± 4.1 × 10⁶ ± 2.2 × 10⁶ ± 1.1 × 10⁷ ± 2.1 × 10⁷ ± 2.2 × 10⁶6.8 × 10⁵ 2.1 × 10⁴ 2.2 × 10⁵ 9.8 × 10⁵ 2.7 × 10⁵ 9.0 × 10⁵ 72 5.6 × 10⁵± 7.3 × 10⁵ ± 1.0 × 10⁶ ± 2.6 × 10⁶ ± 1.3 × 10⁶ ± 5.1 × 10⁶ ± 8.5 × 10⁶± 1.4 × 10⁶ 2.3 × 10⁵ 7.4 × 10⁴ 2.2 × 10⁵ 2.5 × 10⁵ 2.2 × 10⁵ 4.0 × 10⁵

D. Plaque Size Following Viral Infection of GI101A Cells

GLV-1h68, GLV-1h73 and GLV-1h74 were tested for plaque formation inGI101A cells. GLV-1h73 consistently formed larger plaques in GI101Acells than GLV-1h68 did. GLV-1h74 also consistently formed largerplaques in GI101A cells compared to GLV-1h68. This data is consistentwith the higher viral yields of the GLV-1h73 and GLV-1h74 strains ascompared to GLV-1h68.

E. Comparison of Virion Type Produced by Strains GLV-1h68 and GLV-1i69

Vaccinia virus makes three forms of infectious virions during its lifecycle: IMV (intracellular enveloped virus), CEV (cell-associatedenveloped virus) and EEV (extracellular enveloped virus). IMVs are madein virus factories within the infected cells and stay there until celllysis. CEVs have one additional membrane compared to IMVs and areretained on the cell surface. EEVs have identical structures to CEV, butare dissociated from the cell. IMVs are very stable virions, which areimportant for virus transmission between hosts. CEVs are required forefficient cell-to-cell spread. EEVs mediate long-range virustransmission and are relatively resistant to host immune reactions.Because EEVs can be more resistant to host immune attacks than IMVs,EEVs should better survive transit from an initial delivery site to atumor in animals and humans than IMVs, and hence have can haveadvantages as a therapeutic agent for cancer therapy not possessed byIMVs.

The relative levels of cell-associated virus (includes IMV plus CEVforms) and EEV were assessed for strains GLV-1h68 and GLV-1i69 bystandard plaque assay (Table 9). 5×10⁵ CV-1 cells were infected intriplicate with each virus at m.o.i. of 10 and the supernatant (EEV) andinfected cells (cell-associated virus) were harvested 24 hours postinfection. Both EEV and cell-associated virus were titrated in CV-1cells using standard protocols. Viral yields of cell-associated virusversus extracellular enveloped virus (EEV) are shown in Table 9.

Most vaccinia virus strains, including GLV-1h68, produce a majority ofIMVs whereas EEVs only represent a very small portion of virions madeduring infection. The VV A34R gene product is involved in the release ofcell-associated enveloped virus (CEV) from infected cell membranes toform EEV. The proteins encoded by the A34R genes of the GLV-1h68 and WRVV strains have identical amino acid sequences, whereas the proteinsencoded by the A34R genes of the WR (or GLV-1h68) and IHD-J strainsdiffer by two amino acids (Asp 110 (GLV-1h68)→Asn (1HD-J) and Lys 151(GLV-1h68)→Glu (1HD-J); compare SEQ ID NO: 61 and SEQ ID NO: 58). One ofthe mutations, Lys 151 (WR)→Glu (1HD-J) was shown to enhance the releaseof EEV (Blasco et al., J. Virol., 67, 3319-3325, 1993). GLV-1i69 is aderivative of GLV-1h68, in which the GLV-1h68 A34R gene coding sequence(nucleotides 153693 to 154199 of SEQ ID NO: 1) was replaced with theA34R gene coding sequence from vaccinia virus IHD-J strain (SEQ ID NO:58). Vaccinia virus IHD-J produced up to 40 times more extracellularenveloped virus (EEV) than did VV WR strain (Blasco et al., J. Virol.,67, 3319-3325, 1993) and GLV-1h68 produced 8 times as many EEVs asGLV-1h68 did, while both GLV-1i69 and GLV-1h68 viruses made a similaramount of cell associated viruses (i.e. IMV plus CEV) 24 hours postinfection. GLV-1i69 formed comet-like plaques under liquid medium invitro as a result of A34R gene coding sequence replacement, whereasGLV-1h68 generated sharply defined round plaques, indicating thatGLV-1i69 spread faster than GLV-1h68 in vitro. GLV-1i69 thus exhibitsenhanced spreading capability, a characteristic desired in a therapeuticagent for cancer virotherapy, and also can serve as a better source ofEEVs than GLV-1h68.

TABLE 9 Yields of EEVs and cell associated viruses of GLV-1h68 andGLV-1i69 in CV-1 cells Virus Titer (PFU/10⁶ cells) GLV-1h68, cellassociated virus 1.2 × 10⁷ ± 1.8 × 10⁶ GLV-1i69, cell associated virus1.1 × 10⁷ ± 1.5 × 10⁶ GLV-1h68, EEV 7.4 × 10⁴ ± 2.2 × 10³ GLV-1i69, EEV5.8 × 10⁵ ± 5 × 10⁴

Example 3 In Vivo Viral Distribution

A. In Vivo Virus Distribution in Nude Mice with Human Breast TumorXenografts

The ability of the vaccinia viral strains to accumulate in tumor tissuerelative to other tissues was assessed by infecting nude mice that wereimplanted with breast cancer cells in order to form tumors. 5×10⁶GI-101A cells in 100 μl of PBS were injected s.c. into the right lateralthigh of female nude mice, 5 weeks of age, and allowed to grow for 33days. Groups of 4 mice (for each mutant virus strain) were infected viainjection into the femoral vein with 5×10⁶ PFU in 100 μl of PBS ofGLV-1h22, GLV-1h68, GLV-1h70, GLV-1h71, GLV-1h72, GLV-1h73, or GLV-1h74. Two weeks post injection, all mice from each group were sacrificed,and tissue samples were homogenized using MagNA lyser (RocheDiagnostics, Indianapolis, Ind.) at speed of 6,500 for 30 seconds. Theviral titers were determined in duplicate by the standard plaque assayusing CV-1 cells. Results of virus tissue distribution are shown inTable 10 below.

Ovary: No virus was found in the ovaries of the mice infected withGLV-1h22, GLV-1h68, GLV-1h70, GLV-1h71 or GLV-1h73. A moderate amount ofviruses were found in the ovaries of one out of a total of 4 miceinfected with GLV-1h72 or GLV-1h74.

Bladder: No virus was found in the bladders of the mice infected withGLV-1h22, GLV-1h68, GLV-1h71, GLV-1h72 or GLV-1h74. A small amount ofviruses were found in the bladders of one out of a total of 4 miceinfected with GLV-1h70 or GLV-1h73.

Kidney: A small to moderate amount of viruses were found in the kidneysof 50% or more of mice infected with GLV-1h70, GLV-1h72, GLV-1h73 orGLV-1h74, with mice infected with GLV-1h74 having highest viral titer inthe kidney; whereas only small amounts of viruses were found in thekidneys of one mouse infected with GLV-1h22, GLV-1h68 or GLV-1h71.

Adrenal Gland: No virus was found in adrenal glands in any of theinfected mice, except for one mouse infected with GLV-1h72.

Spleen: Moderate amounts of virus particles were found in spleens of allmice infected with GLV-1h68, GLV-1h70, GLV-1h72, GLV-1h73, or GLV-1h74.A smaller amount of viruses were found in spleens of only three out of 4mice infected with GLV-1h71, and one out of four mice infected withGLV-1h22.

Pancreas: No virus was found in the pancreases of GLV-1h22, GLV-1h71,GLV-1h72, and GLV-1h73 infected mice, and only small amounts of viruseswere found in pancreases of two mice infected with GLV-1h74, and onemouse each infected with GLV-1h68 or GLV-1h70.

Lung: Moderate amounts of viruses were found in lungs of all infectedmice. Mice infected with GLV-1h74 exhibited the highest viral titer,whereas mice infected with GLV-1h71 had the lowest viral titer in thelung.

Heart: Moderate amounts of viruses were found in hearts of mice infectedwith GLV-1h74, and only small amount of viruses were found in the heartsof two mice each infected with GLV-1h22 or GLV-1h68, and one mouse eachinfected with GLV-1h70, GLV-1h71, GLV-1h72, GLV-1h73 or GLV-1h74.

Brain: No virus was found in brains of any infected mice, except for onemouse infected with GLV-1h70.

Serum: No virus was found in the sera of mice infected with GLV-1h22 orGLV-1h71. Small amounts of viruses were found in sera of 3 mice infectedwith GLV-1h68 or GLV-1h74, two mice infected with GLV-1h70, and onemouse each infected with GLV-1h72 or GLV-1h73.

Liver: No virus was found in the livers of mice infected with GLV-1h68or GLV-1h72. Small to moderate amounts of viruses were found in liversof all mice infected with GLV-1h73 or GLV-1h74, three mice infected withGLV-1h70, and one mouse each infected with GLV-1h22 or GLV-1h71,respectively.

Tumor: A large number of infectious virus particles were found in tumorsof all infected mice, with mice infected with GLV-1h74 having highestviral titer, whereas mice infected with GLV-1h22 had the lowest viraltiter in the tumor.

In summary, the lung and tumor are the only two organs where viruseswere found in all infected mice. Viruses also were found in spleens inmost of the infected mice. Although the titer of GLV-1h71 in tumors wasfive times as high as that of GLV-1h22, both viruses had similar andlower viral titers in most of the organs tested compared with the miceinfected with other viruses. The removal of Ruc-GFP expression cassettefrom GLV-1h68, which yielded GLV-1h71 derivative, increased the viraltiter in tumors and decreased the viral titers in other organs. Theremoval of the gusA expression cassette from GLV-1h68, which yieldedGLV-1h70 derivative, resulted in a large increase in the viral titer inthe liver, more than a two-fold increase in viral titer in spleencompared to GLV-1h68, and a slight increase in viral titer in the tumor.Deletion of the LacZ expression cassette, which yielded GLV-1h72derivative, increased the viral titer in the tumor, but had less impacton the in vivo virus distribution in other organs than the removal ofgusA expression cassette did, although the viral titer of GLV-1h72 inthe kidney was more than two times as high as that of GLV-1h68. Theviral titers of GLV-1h73, in which both gusA and Ruc-GFP expressioncassettes were deleted, were found to be lower than that of GLV-1h70 inmost of the organs except tumors, and were similar to that of GLV-1h68except that the viral titers of GLV-1h73 in kidneys and tumors weresignificantly higher than that of GLV-1h68. The viral titers ofGLV-1h74, in which all three foreign genes (i.e. Ruc-GFP, gusA and LacZexpression cassettes) were deleted, resulted in increases in viraltiters in most of organs tested.

TABLE 10 Virus Distribution in Nude Mice with Human Breast TumorXenografts 2 weeks post-injection, PFU/g for tissue or PFU/ml for serumOrgan GLV-1h22 GLV-1h68 GLV-1h70 GLV-1h71 GLV-1h72 GLV-1h73 GLV-1h74Ovary 0 0 0 0 2.1 × 10⁴ ± 0 3.8 × 10³ ± 3.5 × 10⁴ 6.6 × 10³ (1)* (1)*Bladder 0 0 6.8 × 10² ± 0 0 3.3 × 10² ± 0 1.2 × 10³ 5.6 × 10² (1)* (1)*Kidney 35 ± 60 1.6 × 10² ± 94 ± 56 31 ± 53 3.9 × 10² ± 66 ± 66 2.8 × 10³± (1)* 2.7 × 10² (3)* (1)* 6.1 × 10² (2)* 4.4 × 10³ (1)* (2)* (3)*Adrenal 0 0 0 0 2.1 × 10⁴ ± 0 0 glands 3.7 × 10⁴ (1)* Spleen 1.7 × 10² ±7.2 × 10² ± 1.7 × 10³ ± 5.9 × 10² ± 7.9 × 10² ± 6.7 × 10² ± 9.4 × 10² ±2.9 × 10² 4.2 × 10² 6.3 × 10² 4.6 × 10² 8.0 × 10² 2.4 × 10² 5.6 × 10²(1)* (4)* (4)* (3)* (4)* (4)* (4)* Pancreas 0 68 ± 120 75 ± 130 0 0 01.7 × 10² ± (1)* (1)* 1.9 × 10² (2)* Lung 6.5 × 10³ ± 9.5 × 10³ ± 1.2 ×10⁴ ± 3.5 × 10³ ± 5.9 × 10³ ± 8.1 × 10³ ± 1.9 × 10⁴ ± 6.2 × 10³ 2.5 ×10³ 3.8 × 10³ 3.3 × 10³ 5.7 × 10³ 4.1 × 10³ 2.3 × 10³ (4)* (4)* (4)*(4)* (4)* (4)* (4)* Heart 1.1 × 10² ± 94 ± 94 54 ± 94 42 ± 73 53 ± 91 50± 86 1.3 × 10³ ± 1.1 × 10² (2)* (1)* (1)* (1)* (1)* 1.1 × 10³ (2)* (4)*Brain 0 0 5.8 × 10² ± 0 0 0 0 1.0 × 10³ (1)* Serum 0 50 ± 35 25 ± 25 013 ± 22 13 ± 22 38 ± 22 (3)* (2)* (1)* (1)* (3)* Liver 24 ± 42 0 1.1 ×10⁴ ± 26 ± 44 0 6.3 × 10³ ± 410 ± 99 (1)* 1.8 × 10⁴ (1)* 1.1 × 10⁴ (4)*(3)* (4)* Tumor 9.4 × 10⁷ ± 3.8 × 10⁸ ± 4.5 × 10⁸ ± 4.7 × 10⁸ ± 7.1 ×10⁸ ± 1.0 × 10⁹ ± 1.3 × 10⁹ ± 9.5 × 10⁷ 3.8 × 10⁸ 3.6 × 10⁸ 2.9 × 10⁸4.1 × 10⁸ 2.4 × 10⁸ 2.5 × 10⁸ (4)* (4)* (4)* (4)* (4)* (4)* (4)*

B. Correlation Between In Vitro and In Vivo Viral Titers

A comparison was made between the in vitro titers of GLV-1h22, GLV-1h68,GLV-1h70, GLV-1h71, GLV-1h72, GLV-1h73, or GLV-1h74 infected GI-101Acells (Example I B) versus the respective in vivo viral titers recoveredfrom the tumor tissues 2 weeks after virus injection (Example 3A). Asummary of the foreign gene insertions in the viral genome of LIVP andtheir effects on in vitro versus in vivo viral titers is shown in Table11. There is a strong correlation between the in vitro and the in vivodata, indicating that the replication of the recombinant VV strains inthe tumors can be well predicted from its replication in the cellcultures.

The viral titers recovered from the tumor tissues also were compared tothe numbers of the inserted expression cassettes that were expressed bythe recombinant virus GLV-1h22, GLV-1h68, GLV-1h70, GLV-1h71, GLV-1h72,GLV-1h73, and GLV-1h74, respectively. There is a strong negativecorrelation between viral titer and the number of inserted expressioncassettes, indicating that when F14.5L, TK, and HA loci were alldisrupted, the greater the number of the foreign gene expressioncassettes that were inserted into these three loci in the viral genome,the greater the strain that was put on virus replication, thus producinga more attenuated virus.

TABLE 11 Comparison of Viral Yields versus Number of HeterologousInserts Virus Yields In vitro In vivo Virus Genotype virus virus VirusF14.5L TK HA yield^(a) yield^(b) Number GLV- pE/L-Ruc- p7.5k- p11k- 5.6× 10⁵ 9.4 × 10⁷ 4 1h22 GFP lacZ gusA pE/L-TFR GLV- pE/L-Ruc- p7.5k-p11k- 7.3 × 10⁵ 3.8 × 10⁸ 3 1h68 GFP lacZ gusA (pE/L- rTFR) GLV-pE/L-Ruc- p7.5k- — 1.0 × 10⁶ 4.5 × 10⁸ 2 1h70 GFP lacZ (pE/L- rTFR) GLV-— p7.5k- p11k- 2.6 × 10⁶ 4.7 × 10⁸ 2 1h71 lacZ gusA (pE/L- rTFR) GLV-pE/L-Ruc- — p11k- 1.3 × 10⁶ 7.1 × 10⁸ 2 1h72 GFP gusA GLV- — p7.5k- —5.1 × 10⁶ 1.0 × 10⁹ 1 1h73 lacZ (pE/L- rTFR) GLV- — — — 8.5 × 10⁶ 1.3 ×10⁹ 0 1h74 ^(a)1.0 × 10⁶ GI-101A cells were infected with each virus atm.o.i. of 0.01 and harvested 3 days PI. ^(b)Viral titers in tumor tissuerecovered 2 weeks post-injection (5 × 10⁶ PFU/mouse, i.v.) from nudemice with implanted GI-101A tumors. ^(c)Only insertions which wereexpressed by the virus are counted.

Example 4 Effects of Modified Viruses on Survival and Tumor Growth InVivo

A. Effects of Viruses Administered to Female Nude Mice on s.c. HumanBreast Tumor Xenografts

Experiment 1

The in vivo effects of GLV-1h22, GLV-1h68, GLV-1h70, GLV-1h71, GLV-1h72,GLV-1h73 and GLV-1h74 were evaluated using a mouse model of breastcancer. Tumors were established in nude mice by subcutaneously injectingG101A human breast carcinoma cells (s.c. on the right lateral thigh;5×10⁶ cells; G101A cells: Rumbaugh-Goodwin Institute for Cancer ResearchInc. Plantation, Fla.; U.S. Pat. No. 5,693,533) into female nude mice(Hsd:Athymic Nude-Foxn1^(nu); Harlan, Indianapolis, Ind.) (n=4-8).Thirty three days following tumor cell implantation, seven groups ofmice (n=3-6) were injected intravenously [in 100 μl of PBS, throughfemoral vein under anesthesia] with 5×10⁶ PFU of GLV-1h22, GLV-1h68,GLV-1 h70, GLV-1h71, GLV-1h72, GLV-1h73 and GLV-1h74, respectively. Thecontrol group of mice was not given any treatment. Tumor volume (mm³)was measured at 33, 36, 43, 50, 57, 64, 71, 78, 82, 85, 89, 92, 97, and102 days post-cancer cell injection. Results of median tumor volume areprovided in Table 12. Each virus provided for a decrease in median tumorvolume relative to uninfected control mice. GLV-1h73 exhibited the besttumor therapy efficacy with a median tumor volume of only 4% that ofuninfected controls after 97 days of tumor growth. GLV-1h70, GLV-1h71,and GLV-1h72 show significantly better tumor therapy efficacy thanGLV-1h68 with median tumor volumes of 20% (GLV-1h70), 19% (GLV-1h71),25% (GLV-1h72), and 33% (GLV-1h68) of that of uninfected controls after97 days of tumor growth. GLV-1h22 could arrest tumor growth overtime;however, during the time period used in this study, tumor growth was notreversed in mice to which GLV-1h22 was administered. GLV-1h74 was ableto reverse tumor growth with high efficacy; however this strain was alsotoxic at this dose and over the extended time period (see Table 12).

TABLE 12 Median tumor volumes at different time points after i.v.injection of different virus strains into nude mice bearing GI-101Atumors Median tumor volume (mm³) Days GLV- GLV- GLV- GLV- GLV- GLV-post- Control 1h22 1h68 1h70 GLV-1h71 1h72 1h73 1h74 implantation (n =3) (n = 6) (n = 5) (n = 4) (n = 6) (n = 4) (n = 6) (n = 6) 33 240.8261.8 248.4 216.8 208.3 157.2 280.3 301.9 36 263.6 273.5 243.8 286.0267.1 155.6 310.9 416.9 43 579.1 536.1 550.4 463.4 543.3 320.1 679.0660.6 50 636.4 701.4 761.3 706.6 721.3 476.3 864.1 828.6 57 671.6 978.4852.0 985.5 936.1 695.0 1117.9 897.4 64 904.3 1203.2 1118.2 1134.11154.1 950.6 1193.6 665.6 71 1235.9 1269.4 1302.0 1147.3 1316.2 1053.6678.6 * 78 1431.8 1437.5 1225.2 1091.2 1069.1 1120.0 373.1 * 82 1888.11537.9 1233.5 1084.7 802.1 1014.8 237.0 * 85 2166.5 1448.5 1295.9 1141.7732.3 1118.0 203.5 * 89 2548.0 1536.1 1083.2 961.3 600.6 842.2 174.3 *92 2715.6 1485.4 1053.6 852.0 606.4 751.8 166.7 * 97 2918.3 1536.9 962.2579.2 546.9 720.1 117.8 * * No median tumor volume was calculated due tothe death of significant numbers of mice.

Experiment 2

In a separate experiment, the in vivo effects of GLV-1h22, GLV-1h68,GLV-1h82, GLV-1h83, GLV-1h84, GLV-1h85 and GLV-1h86 were evaluated usingthe mouse GI-101A breast cancer model. Tumors were established in femalenude mice by s.c. injection 5×10⁶ GI-101A human breast carcinoma cellsinto the right lateral thigh (n=4-8). Thirty eight days following tumorcell implantation, eight groups of mice were injected intravenously with5×10⁶ PFU of GLV-1h22, GLV-1h68, GLV-1 h82, GLV-1h83, GLV-1h84, GLV-1h85and GLV-1h86, respectively, into the femoral vein. Tumor volume (mm³)was measured at 39, 47, 54, 62, 68, 75, and 83 days post-cancer cellinjection. Results of median tumor volume are provided in Table 13.

TABLE 13 Median tumor volumes at different time points after i.v.injection of different virus strains into nude mice bearing GI-101Atumors Days post- implantation Median tumor volume (mm³) of tumor GLV-GLV- GLV- GLV- GLV- GLV- GLV- GLV- cells 1h22 1h68 1h73 1h82 1h83 1h841h85 1h86 39 412.9 350.2 341.1 353.4 392.25 305.6 350.65 419.55 47 750.4722.3 819.8 1081.2 1222.25 604.3 914 962.1 54 1154.3 1301.45 1234.21075.3 1168.75 985.6 1212.8 1279.8 62 1424.4 1390.35 983.2 1319.21686.05 982 947.2 1397.55 68 1849.8 1581.15 855.45 1608.9 2061.5 1119.2636.9 1269.2 75 1907.5 1528.95 517.3 1211.8 1856.35 614.25 255.25 689.683 1973.6 1405.5 172.9 1017.8 1824.35 * * 361.25 89 1887.6 1181.4 74.4855.9 1392.0 * * 187.5 * No median tumor volume was calculated due tothe death of significant numbers of mice.

B. Effects of Viruses on Body Weight and Survival of Tumor-Bearing Mice

1. Post-Infection Survival

The survival rates following i.v. administration of vaccinia strains tonude mice bearing s.c. human breast tumor xenografts were recorded andfound to vary significantly among the different vaccinia strains tested.GLV-1h74 exhibited the highest toxicity with only 17% of mice infectedsurviving the duration of the experiment. In comparison, all miceinfected with GLV-1h22 and GLV-1h71, 67% of mice infected with GLV-1h68and GLV-1h72, and 50% of mice infected with GLV-1h70 and GLV-1h73survived the duration of the experiment. In the case of GLV-1 h74, micestarted to die on day 38 post-infection and most mice died within 48days post-infection. In the GLV-1h68, GLV-1h70, GLV-1h72, and GLV-1h73infections, the first deaths occurred sometime between day 38 and day48, but the death curves were more gradual.

2. Post-Infection Body Weight

The percentage of body weight change following i.v. administration ofthe viruses was also examined and similarly found to vary significantlyamong the different vaccinia strains tested. GLV-1h74 again exhibitedthe most toxicity in mice with a 17% decrease in net body weight 37 daysafter intravenous delivery. GLV-1h22, GLV-1h71, and GLV-1h72 on theother hand, did not elicit any net body weight change in infected mice.GLV-1h68, GLV-1h70, and GLV-1h73 strains did exhibit net body weightchanges in infected mice, though the effects were more gradual withdecreases emerging only after 45 days following infection. At day 65post-infection, mice exhibited decreases in body weight of approximately6%, 6%, and 2.5% for GLV-1h68, GLV-1h70, and GLV-1h73 strainsrespectively.

C. Effects of GLV-1h73 on Body Weight of Mice that Do Not Bear Tumors

Groups of 6-week-old female BALB/c and C57BL/6 mice (7-8 mice per group)were mock-infected (with PBS) or infected via the tail vein with 5×10⁷,1×10⁸ and 2×10⁸ PFU of GLV-1h73. Mice were weighed every two days for 30days and compared with weights on day 1 post infection. Over the courseof the study, both BALB/c and C57BL/6 mice gained more weight at alldoses tested than did the mock-infected mice, indicating no acutetoxicity was caused by GLV-1h73 infection at the dose up to 2×10⁸ PFU.

Example 5 Effect of an Antiviral Agent on Plaque Formation In Vitro

Administration of an antiviral agent to a subject to whom a virus isadministered for tumor treatment can be used to reduce any toxic effectsthat the virus has on the subject. Therefore, the effect of theantiviral agent cidofovir on plaque formation by the recombinantvaccinia virus strains was assessed in vitro by infection of CV-1 cells.Four viruses were tested: GLV-1h68, GLV-1h71, GLV-1h73, and GLV-1h74.CV-1 cells were plated in 24-well plates and were infected with 30PFU/well of each virus for 1 h at 37° C. The inoculum was then removedby aspiration, and 1 ml overlay medium was added per well containing adifferent concentration (in triplicate) of cidofovir (Visitide, GileadSciences, Inc.). The concentrations of cidofovir tested were 0.2, 0.5,2, 5, 20 μg/ml. After incubation in a CO₂ incubator at 37° C. for 48 h,the cells were stained with crystal violet and plaque formation wasassessed.

For all four strains tested, smaller plaques were formed at aconcentration of 5 μg/ml cidofovir. Plaque formation by strains GLV-1h68and GLV-1h71 was almost completely inhibited at 20 μg/ml cidofovir; onlyone tiny plaque for GLY-1h68 and 3 small plaques for GLV-1h71 werefound. For strains GLV-1h73 and GLV-1h74, the number and size of plaqueswere significantly reduced at 20 μg/ml cidofovir, but not totallyinhibited. For all four strains, no significant differences in size ornumber of plaques were seen when control (0 μg/ml cidofovir) experimentswere compared to test experiments in which low levels of cidofovir(i.e., 0.2, 0.5, 2 μg/ml) were used.

Example 6 Effect of an Antiviral Agent on Ability of Modified VacciniaViruses to Arrest or Reverse In Vivo Tumor Growth

The in vivo effect of cidofovir on tumor growth inhibition by modifiedvaccinia virus strain GLV-1h74 was evaluated using a mouse model ofbreast cancer. Tumors were established in nude mice by subcutaneously(s.c.) injecting GI-101A human breast carcinoma cells into female nudemice (see Example 4). Eight mice were tested for each treatment. At 27days after s.c. implantation, the mice were injected with 5×10⁶ PFU ofGLV-1h74 or PBS. Twelve days after virus injection, 0 or three differentdoses (25, 50, or 100 mg/kg, i.p. route) of cidofovir were injected. Allthree doses of cidofovir treatment significantly extended the survivaltime of GLV-1h74 injected mice, indicating attenuation of the viraltoxicity by the cidofovir. The 50 mg/kg dose appeared to work a slightlybetter than the lower 25 mg/kg dose or higher 100 mg/kg dose. Thetreatment with cidofovir did not significantly interfere with tumortherapy by the virus (Table 14). The median tumor volume of the micetreated with virus plus cidofovir was comparable to treatment with virusalone in reversing tumor growth.

TABLE 14 Median tumor volume (mm³) Days post- GLV- GLV- GLV- GI-101A1h74 + 1h74 + 1h74 + tumor cell Untreated GLV-1h74 cidofovir cidofovircidofovir implantation control alone 25 mg/kg 50 mg/kg 100 mg/kg 32204.3 264.9 291.1 279.8 587.1 42 333.9 365.7 314.2 359.5 391.1 50 646.6155.3 238.3 206.5 184.0 56 886.4 (8)* 62.0 (2)* 117.4 (6)* 61.0 (8)*58.9 (6)* *Number of mice surviving at 56 days post tumor cellimplantation

Example 7 Comparison of the Effects on Mouse Body Weight and Survival ofVaccinia Viruses that do not Contain a Functional ThymidineKinase-Encoding Gene A. RVGL2 Vaccinia Strain

The toxicity of a modified vaccinia strain, RVGL2, containing aninsertion of two expression cassettes into the thymidine kinase (TK)gene of strain LIVP was examined in several different mouse tumormodels. Modified vaccinia virus strain RVGL2 was recombinantlyengineered from vaccinia virus LIVP strain (SEQ ID NO: 2). Methods forthe construction of RVGL2 can be found in U.S. Patent Publication No.2005/0031643 (see Example 1 of U.S. Patent Publication No.2005/0031643). RVGL2 contains two marker gene expression cassettes,Ruc-GFP under the control of vaccinia early/late promoter PE/L and lacZunder the vaccinia early promoter P7.5 k, inserted into the TK genecoding sequence. For purposes of comparison, the effects of VV strainsWR and LIVP on body weight and survival also were examined in the samemouse tumor models. Strain WR (ATCC, Manassas, Va.) contains afunctional TK gene. Strain LIVP contains a mutation in the TK gene thatinterrupts the coding sequence and therefore does not encode afunctional thymidine kinase protein.

1. Animal Tumor Models

Athymic nude mice (nu/nu) and C57BL/6 mice (Harlan Animal Res., Inc.,Wilmington, Mass.) at 6-8 weeks of age were used for animal studies.

a. Glioma Model

To establish subcutaneous glioma tumor, rat glioma C6 cells (ATCC No.CCL-107) were collected by trypsinization, and 5×10⁵ cells/0.1 ml/mousewere injected subcutaneously (s.c.) into right hind leg of 6-8 week oldmale athymic mice. On day 7 after C6 cell implantation when median tumorsize was about 150 mm³, viruses at the dose of 10⁷ PFU/0.1 ml/mouse wereinjected intravenously (i.v.) into the femoral vein. Mice weresacrificed 14 days after virus injection.

b. Breast Tumor Model

To develop subcutaneous (s.c) breast tumors in mice, human breast cancerGI-101A cells (Rumbaugh-Goodwin Institute for Cancer Research Inc.Plantation, Fla.; U.S. Pat. No. 5,693,533) at the dose of 5×10⁶cells/0.1 ml/mouse were injected s.c. into the right hind leg of 6-8week old female athymic mice. On day 30 after GI-101A cell implantation,when median tumor size was about 500 mm³, viruses at the dose of 10⁷PFU/mouse were injected i.v. into the femoral vein. Mice were sacrificedon day 14 after virus injection. Mice for survival experiments andbreast tumor therapy studies were kept for long time periods (more than100 days after virus injection). Mice that developed tumors that wereabout 4000 mm³ in size and/or lost 50% of body weight were sacrificed.

c. Melanoma Model

For a melanoma model, mouse melanoma B16-F10 cells (ATCC No. CRL-6475)at the dose of 2×10⁵ cells/0.04 ml/mouse were injected into the foot padof 6-8 week old male C57BL/6 mice. When the tumor was established(median size of tumor about 100 mm³), on day 18 after cell implantation,viruses at the dose of 10⁷ PFU/mouse were injected i.v. into the femoralvein. Mice were sacrificed 10 days after virus injection.

2. Injection of Virus into Animal Tumor Models

VV strains WR, LIVP, and RVGL2, were individually injected i.v. at asingle dose of 1×10⁷ PFU in 100 μl PBS into mice with C6 tumors (7 daysafter implantation of tumor cells), GI-101A tumors (30 days afterimplantation of tumor cells), or B16-F10 tumors (18 days afterimplantation of tumor cells). Body weight was monitored thereafter twicea week. Change of body weight was calculated as follows:((b′−t′)−(b−t))/(b−t), where b and t are the body weight and tumorweight on day of virus injection, and b′ and t′ are the correspondingweights on the day of monitoring (n=4). For measurement of survivalrate, tumorous mice were i.v. injected with individual VV strains at asingle dose of 1×10⁷ PFU/mouse at 30 days after tumor cell implantationand survival was recorded over a 30 to 120-day period, depending on thetumor model.

3. Results

RVGL2 is markedly attenuated and showed significantly lower toxicity inthe mouse tumor models. The survival rate of mice injected with RVGL2 issignificantly longer than mice injected with wild type LIVP or WR. Thedifference in survival of the mice treated with RVGL2 was statisticallysignificant compared with those treated with LIVP or WR (p<0.0001)(n>5). Mice infected with the WR and LIVP strains started to die aroundday 8 or day 20, respectively, after virus infection with no micesurviving past 12 days or 35 days, respectively, after infection. The WRand LIVP infected mice also exhibited weight losses ranging from 15-35%10 days after infection for WR and 5-35% 14 days after infection forLIVP. Mice injected with RVGL2 strain exhibited no weight changes forthe duration of the experiment (up to 14 days after infection) and thedeath curve was more gradual with 100% of the mice surviving up to day80, 70% up to day 105 and 20% at day 120.

The TK gene in the wild-type LIVP is known to be mutated, and nofunctional TK protein is expressed in the infected cells as confirmedthrough a BrdU assay using standard techniques well-known in the art.Because strain RVGL2 is much more attenuated than strain LIVP, yetneither strain encodes a functional TK protein, the attenuation effectis, therefore, not due to loss of TK gene function. The marker proteins,LacZ and Ruc-GFP, contained in the TK locus of RVGL2 also are not knownto have any virus attenuation or tumor therapy function; though theintroduction of the expression cassettes into the TK gene markedlyattenuated the virus.

Example 8 Effects of Route of Administration on In Vivo Models ofAnti-Tumor Therapy

The in vivo effects of vaccinia virus on tumor growth using differentroutes of administration were assessed using the GI-101A mouse breastcancer model. The vaccinia virus strain GLV-1h73 was used for thecomparison. Human breast cancer GI-101A cells at the dose of 5×10⁶cells/0.1 ml/mouse were injected s.c. into the right hind leg of 6-8week old female athymic mice. On day 27 after GI-101A cell implantation,viruses at the dose of 5×10⁶ PFU/mouse were injected using fourdifferent injection methods: intratumoral injection, intravenous tailvein injection, intravenous femoral vein injection, and intraperitonealinjection. Median tumor volume was measured at various time pointsfollowing tumor cell implantation (Table 15).

Following both venous administrations of the virus, the mice exhibitedan initial increase in tumor growth (approximately 3.5-4.5 times thetumor size compared to day of virus injection) followed by a rapidshrinkage of the tumor after 13 days post virus injection with tail veinadministration (40 days post tumor cell implantation in Table 15), and21 days post virus injection with femoral vein administration (47 dayspost tumor cell implantation in Table 15). Tumor eradication wasachieved at approximately 30 days and 50-60 days post virusadministration for the tail vein and femoral vein injections,respectively. For the intratumoral injection of the virus, the miceexhibit less of an initial tumor growth (approximately 2 times the tumorsize compared to day of virus injection); however the eradication of thetumor was much slower than that observed with intravenousadministrations: 80 days compared to 30 or 50-60 days. It also wasobserved that the toxicity of GLV-1h73 when injected into the miceintravenously was higher than the intratumoral injection.Intraperitoneal injection of the virus was unable reverse tumor growth.Taken together, the data suggest that intravenous injection is a morepotent route of administration for eradication of tumors, although thetoxicity of the virus is higher.

TABLE 1 Median tumor volume at different times following injection ofGLV-1h68 via different routes in mice bearing GI-101A tumors Days post-Median tumor volume (mm³) GI-101A Intravenous tumor cell IntravenousFemoral implantation Intratumoral Tail Vein Vein Intraperitoneal 27215.2 388.6 285.6 286.1 33 626.9 972.8 616.9 543.5 40 731.6 1304.31141.0 880.5 48 644.5 650.9 1379.9 1395.4 56 509.6 151.6 813.8 1970.0 63477.5 94.5 609.1 2708.2 69 436.9 74.4 280.4 2867.2 83 264.9 * * 3391.590 261.6 * * 3351.4 104 118.3 * * 3603.6 * No median tumor volume wascalculated due to the death of significant numbers or all of mice.

Example 9 Effect of Combination Therapy with Cisplatin on Ovarian TumorGrowth

The therapeutic effect of an attenuated vaccinia virus alone, or incombination with cisplatin, on the progression of human ovarian tumorswas evaluated in a mouse model of human ovarian cancer. The therapeuticeffect on tumor growth was determined by measuring the volume of anestablished tumor at various time points following administration ofvaccinia virus.

A. Effect of GLV-1h68 on Human Ovarian Tumors

Tumors were established in nude mice by subcutaneously injecting 5×10⁶OVCAR-3 human ovarian carcinoma cells on the right lateral thigh (NIH:OVCAR-3, ATCC No. HTB-161) into female nude mice (Hsd: AthymicNude-Foxn1^(nu); Harlan, Indianapolis, Ind.). Four mice were tested ineach group. Following tumor cell implantation, one group of mice wasinjected with 1×10⁷ PFU/mouse of GLV-1h68 virus in the femoral vein at54 days post-cancer cell injection, whereas the control group wasinjected with phosphate buffered saline (PBS). Tumor volume (mm³) wasmeasured at day 53, day 63, day 69 and day 77. Four tumors were testedat each time point. Results are provided in Table 16.

TABLE 16 Median tumor Days Post- volume (mm³) implantation GLV-1h68Control 0 0.1 0.1 53 176.9 278.1 63 598.8 755 69 668.4 1169.9 77 896.62512.7

Administration of GLV-1h68 virus was able to slow tumor growth, but wasnot able to arrest growth of the OVCAR-3 tumors.

B. Effect of Combination Therapy, GLV-1h68 Plus Cisplatin, on HumanOvarian Tumors

Tumors were established in nude mice by subcutaneously injecting 5×10⁶OVCAR-3 human ovarian carcinoma cells on the right lateral thigh (NIH:OVCAR-3, ATCC No. HTB-161) into female nude mice [Hsd: AthymicNude-Foxn1^(nu); Harlan, Indianapolis, Ind.). Six to eight mice weretested in each group. Following tumor cell implantation, one group ofmice was injected with 2×10⁶ PFUs of GLV-1h68 virus in the tail vein at31 days post-cancer cell injection; one group of mice wasintraperitoneally injected with 5 mg/kg cisplatin once a day on days 51,52, 54 and 55 post-cancer injection; one group received combinationtherapy of GLV-1h68 and cisplatin, and the control group of mice was notgiven any treatment. Tumor volume (mm³) was measured at days 41, 55, 64,71, 79, 87, 93, 99, 106, 113, and 119 post-cancer cell injection.Results are provided in Table 17.

TABLE 17 Median tumor volume (mm3) following treatment GLV-1h68 DaysPost- alone Cisplatin GLV-1h68 + No treatment implantation (n = 7) alone(n = 8) cisplatin (n = 6) (n = 8) 41 298.8 479.5 415.1 348.2 55 1448.31748.5 1403.1 1972.7 64 2512.4 1553.3 1163.7 4969.5 71 3407.4 1297.0993.0 * 79 * 2280.4 757.5 * .87 * 4108.8 667.0 * 93 * * 547.0 * 99 * *549.0 * 106 * * 511.5 * 113 * * 465.3 * 119 * * 441.3 * * No mediantumor volume was calculated due to the death of significant numbers orall of mice.

Treatment with GLV-1h68 virus alone decreased tumor growth rate, but didnot shrink the tumors. Tumors treated with GLV-1h68 virus alone werepartially filled with pus and were purplish in color in some areas ofthe tumor surface compared to untreated animals that are full of pus andpurplish in color overall on the surface. Treatment with cisplatin aloneinitially reversed tumor growth, but two weeks after discontinuedtreatment, the tumors began growing again exponentially. Tumors treatedwith cisplatin alone are similar in appearance to non-treated mice andare full of pus and purplish in color. In the presence of both GLV-1h68virus and cisplatin, tumor shrinkage was sustained until the end-pointof the experiment (i.e., 119 days post-injection of cancer cells).Tumors treated with the combination therapy were solid (with no pus) andwhitish in color, a phenotype characteristic of dying tumors thatundergo significant shrinkage. Thus, the combination therapy was mosteffective in controlling and inhibiting ovarian tumor progression.

C. Effect of Combination Therapy, GLV-1h68 Plus Carboplatin, on HumanOvarian Tumors

Tumors were established in nude mice by subcutaneously injecting 5×10⁶OVCAR-3 human ovarian carcinoma cells on the right lateral thigh (NIH:OVCAR-3, ATCC No. HTB-161) into female nude mice [Hsd: AthymicNude-Foxn1^(nu); Harlan, Indianapolis, Ind.). Six to eight mice weretested in each group. Following tumor cell implantation, one group ofmice was injected with 5×10⁶ PFUs of GLV-1h68 virus in the femoral veinat 56 days post-cancer cell injection; one group of mice wasintraperitoneally injected with 32.5 mg/kg carboplatin in 200 μPBS onday 63, 66, 69, 72, 75, 78, 81 and day 84 post-cancer cell injection fora total of 8 doses; one group received combination therapy of GLV-1h68and cisplatin; and the control group of mice was not given anytreatment. Tumor volume (mm³) was measured at days 55, 62, 70, 75, 81,89 and 96 post-cancer cell injection. Results are provided in Table 17a.

TABLE 17a Median tumor volume (mm³) following treatment Days Post-GLV-1h68 Carboplatin GLV-1h68 + implantation alone alone cisplatin Notreatment 55 268.4 676.45 402.1 793.45 62 623.7 1565.85 1128.15 1778.2570 1232.5 5708.9 1485.05 3261.4 75 1277.05 3119.65 1599.35 4649.5 811189.55 3733.95 1411.4 7198.7 89 774.4 4109.75 1040.15 * 96 4460.5906.75 *

Treatment with GLV-1h68 virus alone or combination therapy with GLV-1h68virus and cisplatin appeared effective in slowing the initial tumorgrowth rate and then shrinking the tumor. Tumor shrinkage in micetreated with either regimen was sustained until the end-point of theexperiment. While monotherapy with cisplatin reduced the rate of tumorgrowth, compared to untreated mice, the treatment was not able to reducethe size of the tumors or arrest growth completely.

Example 10 Comparison of Two Chemotherapeutic Agents in CombinationTherapy Against Human Breast Carcinoma Tumors In Vivo

The therapeutic effect of an attenuated vaccinia virus alone, or incombination with either cisplatin or doxorubicin, on the progression ofhuman breast carcinoma tumors was evaluated in a direct in vivo study.

Tumors were established in nude mice by subcutaneously injecting 5×10⁶cells GI-101A human breast carcinoma cells (Rumbaugh-Goodwin Institutefor Cancer Research Inc. Plantation, Fla.; U.S. Pat. No. 5,693,533]subcutaneously on the right lateral thigh of female nude mice(Hsd:Athymic Nude-Foxn1^(nu); Harlan, Indianapolis, Ind.;n=4-8mice/group). Following tumor cell implantation, one group of micewas injected with 1×10⁶ PFU/mouse of GLV-1h68 virus in the tail vein at32 days post-cancer cell injection, one group of mice wasintraperitoneally injected with 5 mg/kg cisplatin once daily on each ofdays 47, 48, 49, 50 and 51 post-cancer cell injection, one groupreceived combination therapy of GLV-1h68 and cisplatin, one group ofmice was intraperitoneally injected with 3 mg/kg doxorubicin (SigmaCatalog no. 44583) alone once a week for 4 consecutive weeks starting 47days post-cancer cell injection, one group of mice received combinationtherapy of GLV-1h68 and doxorubicin, and the control group of mice wasnot given any treatment. Tumor volume (mm³) was measured at days 32, 47,52, 56, 63, 67, 76, 80, 89, 96 and 104 post-cancer cell injection.Results are provided in Table 18.

TABLE 18 Median tumor volume (mm3) on days post-GI-101A injection GLV-1h68 GLV-1h68 + GLV-1h68 + No Days Post- alone Cisplatin cisplatinDoxorubicin doxorubicin treatment implantation (n = 7) alone (n = 5) (n= 6) alone (n = 5) (n = 4) (n = 8) 32 212.7 208.9 184.4 192.4 155.2171.5 47 694.0 646.0 538.8 664.0 496.9 463.1 52 810.7 622.1 582.4 856.6558.0 561.3 56 901.7 637.7 570.1 968.2 599.1 667.9 63 1096.2 865.1 893.11328.6 850.5 1066.3 67 990.6 963.1 916.4 1390.2 1066.6 1105.7 76 914.31260.6 772.7 1884.1 1296.7 1420.9 80 903.1 1484.3 692.4 2213.1 1308.91959.9 89 801.5 2171.9 669.2 2484.8 1457.1 2948.1 96 644.4 2996.1446.6 * 1357.6 3453.9 104 525.9 2849.4 454.1 * 1339.8 4202.5 * No mediantumor volume was calculated due to the death of significant numbers orall of mice.

In contrast to the OVCAR-3 human ovarian carcinomas, treatment ofGI-101A human breast carcinomas with GLV-1h68 alone resulted in tumorshrinkage. Treatment with cisplatin alone decreased the rate of tumorgrowth, but did not shrink tumors. Treatment with doxorubicin alone didnot have any effect on the rate of tumor growth and results were similarto the untreated control animals. Treatment of animals with acombination of doxorubicin and GLV-1h68 was more effective than theuntreated control animals, but was not as effective as GLV-1h68treatment alone, thus, doxorubicin may inhibit viral oncolytic activity.Treatment of animals with a combination of cisplatin and GLV-1h68 hadthe greatest effect on the shrinkage of the tumors and exhibitedphenotypes characteristic of dying tumors as described above.

Example 11 Effect of Combination Therapy with Cisplatin on HumanPancreatic Tumors

The therapeutic effect of a modified vaccinia virus alone, or incombination with cisplatin, on the progression of human pancreatictumors was evaluated in a direct in vivo study of a mouse model of humanpancreatic cancer. The therapeutic effect on tumor growth was determinedby measuring the volume of the tumor at various time points.

A. Effect of GLV-1h68 or Cisplatin on Human Pancreatic Tumors

Tumors were established in nude mice by subcutaneously injecting 5×10⁶cells PANC-1 human pancreatic carcinoma cells (ATCC No. CRL-1469)subcutaneously on right lateral thigh of male nude mice (Hsd:AthymicNude-Foxn1^(nu); Harlan, Indianapolis, Ind.; n=3-8 mice/group).Following tumor cell implantation, one group of mice was injected with2×10⁶ PFU/mouse of GLV-1h68 virus in the tail vein 37 days post-cancercell injection, one group of mice was intraperitoneally injected with 5mg/kg cisplatin on each of days 46, 47, 48, 49 and 50 post-cancer cellinjection, and the control group of mice was not given any treatment.Tumor volume (mm³) was measured at days 36, 46, 52, 57, 63, 72, 79, 86,94 and 100 post-cancer cell injection. Results are provided in Table 19.

TABLE 19 Median tumor volume (mm³) No treatment GLV-1h68 Cisplatin DaysPost- (n = 6) (n = 8) (n = 3) 36 196.2 153.1 170.2 46 300.9 351.8 296.652 400.8 385.3 254.95 57 540.2 396.3 326.2 63 721.4 247.7 460.3 721082.5 156.8 663.4 79 1640.3 128.4 1022.0 86 2599.8 64.5 1718.3 943927.9 56.6 2520.4 100 4556.7 39.5 3254.1

Although cisplatin slowed the growth rate of the pancreatic tumorsignificantly compared to untreated controls, it was unable to arresttumor growth. GLV-1h68, on the other hand, caused shrinkage of thepancreatic tumors as early as 29 days after virus injection.

B. Effect of Combination Therapy, GLV-1h68 with Cisplatin, on PANC-1Human Pancreatic Tumors

Tumors were established in nude mice by subcutaneously injecting 5×10⁶cells PANC-1 human pancreatic carcinoma cells (ATCC# CRL-1469)subcutaneously on right lateral thigh of male nude mice (n=8-11mice/group). Following tumor cell implantation, one group of mice wasinjected with 1×10⁶ PFU/mouse of GLV-1h68 (RVGL21) virus in the tailvein 32 days post-cancer cell injection, one group of mice was treatedwith a combination of GLV-1h68 (1×10⁶ PFU/mouse of GLV-1h68 injected inthe tail vein at day 32) and intraperitoneal injection of 6 mg/kgcisplatin once daily on each of days 42, 43, 44, 45 and 46), one groupreceived cisplatin only, and the control group received no treatment.Tumor volume (mm³) in mice administered GLV-1h68 or GLV1h68 andcisplatin was measured at days 31, 46, 52, 59, 68, 75, 84, 90 and 96post-cancer cell injection. Tumor volume (mm³) in the control group andmice administered cisplatin only was measured at days 36, 46, 52, 57,63, 72 and 79 days post-cancer cell injection. Results are provided inTable 20.

TABLE 20 Median tumor volume at different times following treatment withGLV-1h68 and cisplatin in mice bearing PANC-1 tumors Median tumor volume(mm³) Days Post- GLV-1h68 GLV-1h68 + No implantation (n = 11) Cisplatin(n = 8) Cisplatin treatment 31 (36) 118.9 125.8 170.2 196.15 46 (46)282.6 365.6 296.6 300.9 52 (52) 315.9 206.5 254.95 400.8 59 (57) 291.6325.4 326.2 540.2 68 (63) 290.5 250.8 460.3 721.35 75 (72) 209.5 122.0663.4 1082.45 84 (79) 196.8 70.7 1022 90 119.9 51.9 96 133.5 0

Tumor shrinkage was more pronounced with the combination therapy ofGLV-1h68 in combination with cisplatin compared to GLV-1h68 alone.Tumors were resolved with the combination therapy on day 64 post-virusinjection.

C. Effect of Combination Therapy, GLV-1h68 with Cisplatin, on MIA-PaCa2Human Pancreatic Tumors

The effect of combination treatment with GLV-1h68 and cisplatin wasevaluated using a second mouse model of human pancreatic cancer. Tumorswere established in nude mice by subcutaneously injecting 5×10⁶ cellsMIA PaCa-2 human pancreatic carcinoma cells (ATCC No. CRL-1420)subcutaneously on right lateral thigh of male nude mice (Hsd:AthymicNude-Foxn1nu; Harlan, Indianapolis, Ind.; n=3-8 mice/group). Thirty-onedays following tumor cell implantation, mice in on group were injectedintravenously [in 100 μl of PBS, through femoral vein under anesthesia]with 5×10⁶ PFU of GLV-1h68, followed by intraperitoneal injection of 4mg/kg cisplatin on day 42, 43, 44, 45 and 46; mice in another group wereinjected intravenously with 5×10⁶ PFU of GLV-1h68; mice in a furthergroup were injected intraperitoneally with 4 mg/kg cisplatin on day 42,43, 44, 45 and 46; and mice in the control group received no treatment.Tumor volume (mm³) was measured at 31, 42, 48, 56, 64 and 70 dayspost-cancer cell injection. Results of median tumor volume (mm³) areprovided in Table 20a.

Combination therapy of GLV-1h68 in combination with cisplatin andvirotherapy with GLV-1h68 alone effectively controlled tumor growthcompared to monotherapy with cisplatin or no treatment. Combinationtherapy of GLV-1h68 in combination with cisplatin and virotherapy withGLV-1h68 alone appeared equally effective at shrinking and controllingMIA-PaCa2 tumors in nude mice.

TABLE 20a Median tumor volume at different times following treatmentwith GLV-1h68 and cisplatin in mice bearing MIA-PaCa2 tumors Mediantumor volume (mm³) Days Post- GLV-1h68 + implantation No treatmentGLV-1h68 cisplatin cisplatin 31 681.45 537.9 577.7 674.9 42 3295.052110.9 2991.25 2195.8 48 4164.3 1916.1 5171.9 1741.75 56 5586.1 14628454.65 1623.35 64 * 1191 * 1321.85 70 * 1200.3 * 1130.35

Example 12 Effect of Combination Therapy with Gemcitabine on HumanTumors

The therapeutic effect of a modified vaccinia virus alone, or incombination with gemcitabine, on the progression of human lung tumorsand human pancreatic tumors was evaluated in vivo mouse tumor models.

A. Effect of Combination Therapy, GLV-1h68 with Gemcitabine, on HumanLung Carcinoma Tumors

The therapeutic effect of a modified vaccinia virus alone, or incombination with gemcitabine, on tumor growth inhibition was evaluatedin a direct in vivo study in a mouse model of human lung cancer. Thetherapeutic effect on tumor growth was determined by observingphenotypic changes in the tumors and by measuring the volume of thetumor at various time points.

Tumors were established by subcutaneously injecting A549 human lungcarcinoma cells [s.c. on right lateral thigh; 5×10⁶ cells; ATCC#CCL-185] into male nude mice [Hsd:Athymic Nude-Foxn1^(nu); Harlan,Indianapolis, Ind.] (n=4-8). On day 23 after A549 cell implantation,viruses at a dose of 5.0×10⁶ PFU/mouse were injected intravenously(i.v.) into the femoral vein. At 7, 10, 13, 16 and 19 days after virusinjection, Gemcitabine (Gemzar®, Eli Lilly and Company, at 50 mg/kg or100 mg/kg) was injected intraperitoneally (i.p.). Median tumor volume(mm³) was measured at 22, 30, 37, 44, 51, 58, and 65 days post-tumorcell implantation. Results are provided in Table 21a.

Gemcitabine alone actually increased tumor growth in the mice at both 50mg/kg and 100 mg/kg doses. In contrast, gemcitabine enhanced the slowingof tumor growth and tumor shrinkage of GLV-1h68 when administered in thecombination with GLV-1h68 at earlier time points followingadministration of the virus (compare e.g., day 37 post-tumor cellimplantation onward). Combination therapy with the lower dosage (50mg/kg) of gemcitabine promoted a more rapid response of tumor shrinkageas compared to the higher dosage or GLV-1h68 alone.

TABLE 21a Effect of Gemcitabine Combination Therapy on Human Lung TumorsMedian tumor volume (mm³) GLV-1h68 + GLV-1h68 + Days Post- No GLV-Gemcitabine Gemcitabine Gemcitabine Gemcitabine implantation treatment1h68 50 mg/kg 50 mg/kg 100 mg/kg 100 mg/kg 22 226.5 303.8 245.6 234.0228.1 251.9 30 477.4 677.2 579.8 565.7 487.4 599.1 37 557.1 1031.0 745.3725.5 693.6 906.5 44 870.0 885.7 1023.9 544.4 1046.0 796.8 51 1442.7902.0 1229.7 485.6 1580.2 616.3 58 1520.4 456.8 1619.0 336.9 2216.6444.9 65 2168.7 262.1 2852.0 393.5 3413.4 424.1B. Effect of Combination Therapy, GLV-1h68 with Gemcitabine, on HumanPancreatic Tumors

The therapeutic effect of a modified vaccinia virus alone, or incombination with gemcitabine, on tumor growth inhibition was evaluatedin a direct in vivo study in a mouse model of human pancreatic cancer.The therapeutic effect on tumor growth was determined by observingphenotypic changes in the tumors and by measuring the volume of thetumor at various time points.

Tumors were established by subcutaneously injecting PANC-1 humanpancreatic carcinoma cells [s.c. on right lateral thigh; 5×10⁶ cells;ATCC# CRL-1469] into male nude mice [Hsd:Athymic Nude-Foxn1^(nu);Harlan, Indianapolis, Ind.] (n=3-8 mice/group). On day 28 afterPANC-1cell implantation, viruses at a dose of 5.0×10⁶ PFU/mouse wereinjected intravenously (i.v.) into the tail vein. At 7, 10, 13, 16 and19 days after virus injection, Gemcitabine (Gemzar®, Eli Lilly andCompany, at 50 mg/kg or 100 mg/kg) was injected intraperitoneally(i.p.). Median tumor volume (mm³) was measured at 28, 35, 42, 50, 56,63, 71 and 79 days post-tumor cell implantation. Results are provided inTable 21b.

Gemcitabine alone moderately decreased tumor growth in the mice at both50 mg/kg and 100 mg/kg. In combination with GLV-1h68, gemcitabineenhanced the slowing of tumor growth and promoted tumor shrinkage byGLV-1h68 (compare e.g., day 42 post-tumor cell implantation onward).Combination therapy with the lower dosage (50 mg/kg) of gemcitabinepromoted a more rapid response of tumor shrinkage as compared to thehigher dosage or GLV-1h68 alone.

TABLE 21b Effect of Gemcitabine Combination Therapy on Human PancreaticTumors Median tumor volume (mm³) GLV-1h68 + GLV-1h68 + Days Post- NoGLV- Gemcitabine Gemcitabine Gemcitabine Gemcitabine implantationtreatment 1h68 50 mg/kg 50 mg/kg 100 mg/kg 100 mg/kg 28 281.6 231.7268.0 226.2 209.7 227.8 35 395.4 425.9 359.2 408.6 417.8 401.3 42 616.7724.5 543.6 592.3 652.5 662.5 50 1114.6 845.1 584.8 513.2 810.3 569.7 561146.5 776.3 682.9 510.1 655.6 554.1 63 1446.1 547.9 654.7 372.5 407.3508.8 71 2074.6 376.3 1006.2 257.9 789.1 281.6 79 2907.7 268.2 1534.1257.7 1437.0 242.4

Example 13 Effect of Vaccinia Virus Expression of Human Plasminogen k5Domain on Human Lung Carcinoma Tumors

The therapeutic effect of administration vaccinia viruses expressing anangiogenesis inhibitor on the progression of A549 tumors was evaluatedin a direct in vivo study in a mouse model of human lung cancer.GLV-1h81, which expresses human plasminogen k5 domain, and the GLV-1h68control strain were used for the study. The therapeutic effect on tumorgrowth was determined by observing phenotypic changes in the tumors andby measuring the volume of the tumor at various time points.

Tumors were established by subcutaneously injecting A549 human lungcarcinoma cells [s.c. on right lateral thigh; 5×10⁶ cells; ATCC#CCL-185] into male nude mice [Hsd:Athymic Nude-Foxn1^(nu); Harlan,Indianapolis, Ind.] (n=4-8). On day 23 after A549 cell implantation,viruses at a dose of 5.0×10⁶ PFU/mouse were injected i.v. into thefemoral vein. Median tumor volume (mm³) was measured at 22, 30, 37, 44,51, 58, and 65 days post-tumor cell implantation. Results are providedin Table 22. In the mouse model for human lung carcinoma, GLV-1h81,which expresses, human plasminogen k5 domain, was able to slow tumorgrowth though the effect was less pronounced than the GLV-1h68 strain.The difference in therapeutic effect may be due to an attenuating effectof the strong synthetic early/late promoter on the GLV-1h81 virus.

TABLE 22 Days Median tumor volume (mm³) Post- No implantation treatmentGLV-1h68 GLV-1h81 22 226.5 303.8 290.9 30 477.4 677.2 651.2 37 557.11031.0 955.6 44 870.0 885.7 1087.7 51 1442.7 902.0 1068.5 58 1520.4456.8 756.0 65 2168.7 262.1 685.9

Example 14 Imaging of Viruses Expressing Multiple Proteins for Detection

Recombinant vaccinia viruses that express click beetle luciferase-mRFP I(CBG99-mRFP1) and Renilla luciferase-GFP (Ruc-GFP) fusion genes weregenerated as described above to facilitate evaluation of virusreplication in vitro and monitoring virus therapeutic effects and spreadin vivo. GLV-1h84, which expresses CBG99-mRFP1, and GLV-1h86, whichexpresses Ruc-GFP, were used to evaluate the ability to monitor virusesin vitro and in vivo by fluorescence and bioluminescence imagingtechniques. In the GLV-1h84 strain, CBG99 and mRFP1 are connectedthrough a picornavirus 2A element. During translation, these twoproteins are cleaved into two individual proteins at picornavirus 2Aelement. In the GLV-1h86 strain, Ruc-GFP is expressed as a fusionprotein. As described in Example 1, the GLV-1h84 strain exhibits strongexpression of mRFP1 as confirmed by fluorescence microscopy.

Real-time monitoring of infection was performed using CV-1 (green monkeykidney cells) and GI-101A (human breast adenocarcinoma) cells infectedwith GLV-1h84 and GLV-1h86. Infection of CV-1 and GI-101A cells withGLV-1h84 at an m.o.i. of 0.01 was monitored in real-time usingfluorescence microscopy. In both cell types, individual red plaques wereseen 24 hours post infection (hpi). By 48 hpi, more than 80% of cellswere infected; at 72 hpi, almost all cells were infected. GLV-1h86showed similar spreading patterns in both CV1 and GI-101A cells incomparison with GLV-1h84. Infection of both cell types with eitherGLV-1h84 or GLV-1h86 was also imaged by measuring luciferase activities.

In a separate experiment of in vitro infection of CV1 cells, luciferaseassays of 0.5 mg cell extract were performed at different times postinfection. GLV-1h68 Ruc-GFP infected CV1 cells were assayed with 0.0375mg coelenterazine and GLV-1h84 CBG99-RFP infected CV1 cells with 0.5 mgbeetle luciferin. Even though the click beetle luciferase was incubatedwith more luciferin, the photon emission during the assay was comparablefor both luciferases. Data is shown in Table 23.

TABLE 23 Time Hours Relative Light Units Post- GLV1h68 GLV-1h84Infection Ruc-GFP CBG99-RFP 0 16 26 2 22 52 4 173 402 6 22295 19824 810874 11070 10 45550 52618 12 95445 109189 14 214488 235280 18 661022778738 24 14595754 15082362 32 3095943 3436978 40 2047472 2265043 481641432 1720210

Real-time monitoring of co-infection of the two viruses was alsoperformed using CV-1 and GI-101A cells infected with both GLV-1h84 andGLV-1h86. When CV-1 cells were co-infected with GLV-1h84 and GLV-1h86 atan m.o.i. of 0.01 for each virus, individual red or green plaques wereseen at 24 hpi under a fluorescence microscope, confirming that eachvirus plaque was derived from a single virus particle. At 48 hpi, mostcells were infected. At later hpi, some of infected cells were yellow,indicating that cells infected with one virus can also be infected laterwith another virus. By 72 hpi, most cells were co-infected. In contrast,when GI-101A cells were co-infected with both viruses, there were notmany yellow cells even by the time of 72 hpi, suggesting that the virusdoes not spread very well to GI-101A cells already infected.

Flow cytometric analysis was also performed on CV-1 cells infected witheither GLV-1h84 or GLV-1h86 viruses or a combination of the two virusesto determine the levels of fluorescent molecule expression as well asassess any interference in the replication of the viruses following dualinfection of viruses. CV-1 cells were infected at a m.o.i. of 0.5 forthe single virus strain infection and a m.o.i. of 0.25 for each virusfor the mixed infection. No significant differences were seen betweenthe VV strains in the replication efficiency of the viruses. Expressionof GFP was detectable earlier than RFP. Vv strains did not influencefluorescence expression on each other as can be seen from the comparisonof expected fluorescent cells and GFP and RFP expressing cells in themixed infection. Data for the percentage of fluorescent cells for eachinfection is presented in Table 24.

TABLE 24 Percentage of Fluorescent Cells GFP + Total Expected Hours GFPin RFP in RFP in fluorescence GFP + RFP Post mixed mixed mixed in mixedin mixed Infection GFP RFP infection infection infection infectioninfection 0 0.07 0.07 0.07 0.07 0.07 0.07 0.000049 4 0.27 0.07 0.11 0.120.08 0.15 0.000132 8 3.29 0.12 2.68 0.10 0.06 2.72 0.002 12 18.38 9.8812.23 4.09 1.80 14.52 0.500 24 56.01 56.37 42.32 42.87 22.02 63.1718.142 48 88.12 97.6 60.26 75.02 43.00 92.28 45.207

In vivo imaging was also performed by infecting tumor bearing mice withCBG99-mRFP1 expressing viruses. C6(pLEIN) glioma tumor bearing nude micewere injected intravenously with 5×10⁶ PFU of GLV-1h84 and imaged at 7days post infection. Tumor and poxes on the tail were detected byfluorescence and bioluminescence imaging of mRFP1 and CBG99,respectively. In another experiment GI-101A tumor bearing nude mice wereinjected with GLV-1h84 CBG99-RFP or Ruc-GFP expressing virus and imagedat 7 days p.i. Luciferase activity indicating the presence of the viruswas detected in vivo at the location of the tumor following injection ofclick beetle luciferin or coelenterazine into the mice and detection bylow light imaging methods. Detection of fluorescence emission by eitherRFP or GFP was also detected in vivo in live mice as well as in excisedtumors.

The results of these studies show that vaccinia virus replication incultured cells and in living mice can be monitored by both fluorescenceand bioluminescence imaging. In addition to their use as diagnostictools, these strains can be used to investigate the role of the immunesystem and pathogen clearance in initial tumor colonization. Thedescribed VV strains are useful tools to investigate the influence ofone Vaccinia infection followed by a second infection since they did notinfluence each other in their replication but were clearlydistinguishable from each other due to their multicolor labeling. Onevirus expresses GFP and Renilla luciferase (Ruc) and can be detected byfluorescence imaging at emission wavelength 509 nm for GFP and by photonemission at 482 nm after adding coelenterazine for Ruc; the other VVexpresses RFP and click beetle luciferase (CBG99) and can be detected byfluorescence imaging at emission wavelength 583 nm for RFP and by photonemission at 537 nm after adding beetle luciferin for CBG99. Hence, bothviruses can be used independently in the same mouse for comparative lowlight imaging and high resolution in in vivo and ex vivo histologyanalysis.

Example 15 Resonance Imaging of Viruses Expressing Iron Binding Proteins

Expression of iron binding proteins can enhance the imaging propertiesof viruses for in vivo detection. Vaccinia viral strains expressing theiron binding proteins, such as a ferritin and a transferrin receptorwere tested for the ability to be detected in vivo using magneticresonance imaging (MRI). Three strains were tested (GLV-1h22, GLV-1h82,and GLV-1h83) and compared to a control strain (GLV-1 h68) that does notexpress the iron binding proteins. GLV-1h22 expresses the transferrinreceptor, GLV-1h82 expresses both the transferrin receptor and E. coliferritin, and GLV-1h83 expresses E. coli ferritin.

Tumors were established in athymic nu-/nu-mice by subcutaneouslyinjecting 5×10⁶ cells GI-101A human breast carcinoma cellssubcutaneously on the right lateral thigh of female nude mice. At 30days post tumor cell implantation, mice were i.v. injected withdifferent vaccinia virus strains or PBS control into the lateral tailvein. At 14 days later (44 days post tumor cell implantation), mice wereperfused using 4% formaldehyde. Colonization of VV was confirmed by GFPexpression in the tumor. Tumors were then excised and MRI was performed(Spin echo sequence TR: 1200 ms, TE: 35 ms, rat coil (UCSD) 7T GE smallanimal MRI scanner). The resulting pictures were analyzed and the meangrey level of each tumor was determined. The results for the grey levelsare shown in Table 25. Expression of the ferritin or the transferrinreceptor enhanced the MRI contrast in the tumor tissue compared to theuninfected and GLV-1h68 controls. The co-expression of ferritin with thetransferrin receptor, however, did not increase the effect. Expressionof ferritin alone appeared to have the greatest effect, which suggeststhat there may be an attenuating effect on gene expression whenadditional expression cassettes are added to the virus or aninterference effect of expressing a human transferrin receptor in amouse cell. Nonetheless the experiments establish that expression ofiron binding proteins or iron transporters is useful for detection oftumors.

TABLE 25 Grey Standard Level Deviation Mean PBS control 111 33 108(uninfected) GLV-1h68 100 26 99 GLV-1h22 (hTfR) 85 25 83 GLV-1h82 a(ftn, hTfR) 85 25 85 GLV-1h82 b (ftn, hTfR) 83 28 81 GLV-1h83 (ftn) 7430 73

Example 16 Effects of Modified Viruses on Lung Tumor Growth In Vivo A.Effects of Viruses Administered to Male Nude Mice on Human Lung Tumors

Tumors were established by subcutaneously injecting A549 human lungcarcinoma cells [s.c. on right lateral thigh; 5×10⁶ cells; ATCC#CCL-185] into male nude mice [Hsd:Athymic Nude-Foxn1^(nu); Harlan,Indianapolis, Ind.] (n=4-8). On day 23 after A549 cell implantation,GLV-1h68, GLV-1h71, GLV-1h72 and GLV-1h73 viruses at a dose of 5×10⁶PFU/mouse were injected i.v. into the femoral vein. Median tumor volume(mm³) was measured at time points post-tumor cell implantation.

All three strains GLV-1h68, GLV-1h72, and GLV-1h73 promoted rapidresponses of tumor shrinkage. The tumor shrinkage response induced byGLV-1h72 and GLV-1h73 was slightly faster. Treatment with GLV-1h71 ledto approximately 50% decrease in tumor growth, but did not result incomplete reverse of tumor growth as seen in the treatment groups ofother three viruses.

TABLE 26 Median tumor volumes at different time points after i.v.injection of different virus strains into nude mice bearing A549 tumorsDays post- implantation of Median tumor volume (mm³) tumor No GLV- GLV-GLV- GLV- cells Treatment 1h68 1h71 1h72 1h73 16 126.0 170.6 148.9 135.8140.5 22 268.0 294.9 338.3 342.3 362.7 29 518.3 683.4 568.1 604 575 36768.9 882.7 766.4 709.7 663.2 44 1004.1 586.0 802.4 418.85 398.1 511322.4 283.6 783.9 263.9 271.6 57 1913.0 * 897.6 177 210.5

Example 17 Effects of A35R Deletion on Virulence and Tumor Growth InVivo

A. Effects of Modified Viruses Administered to Female Nude Mice on s.c.Human Breast Tumor Xenografts

The in vivo effects of removal of the A35R gene on virulence and tumorshrinkage induced by the modified vaccinia strains were evaluated usinga mouse model of breast cancer. Strains GLV-1h68, GLV-1h73 and GLV-1h74were evaluated with their corresponding A35R-deleted strains GLV-1j87,GLV-1j88 and GLV-1j89, respectively. Tumors were established in nudemice by subcutaneously injecting GI-101A human breast carcinoma cells(s.c. on the right lateral thigh; 5×10⁶ cells; GI-101A cells:Rumbaugh-Goodwin Institute for Cancer Research Inc. Plantation, Fla.;U.S. Pat. No. 5,693,533) into female nude mice (Hsd:AthymicNude-Foxn1^(nu); Harlan, Indianapolis, Ind.) (n=4-8). Thirty three daysfollowing tumor cell implantation, groups of mice were injectedintravenously [in 100 μl of PBS, through femoral vein under anesthesia]with 5×10⁶ PFU of GLV-1h68, GLV-1h73, GLV-1 h74, GLV-1j87, GLV-1j88 andGLV-1j89, respectively. The control group of mice was not given anytreatment. Tumor volume (mm³) was measured at 34, 41, 49, 57, 64, 71,78, 85 and 92 days post-cancer cell injection. Results of median tumorvolume are provided in Table 27.

GLV-1h73 and GLV-1h74 showed antitumor activities similar to theircorresponding A35R-deleted strains, GLV-1j88 and GLV-1j89, respectively.A35R-deleted strain GLV-1j87 showed significantly enhanced antitumoractivity as compare to its corresponding strain of GLV-1h68. The A35Rdeletion was able to attenuate the toxicity of the GLV-1h68 virus andprovide a greater tumor response (see GLV-1j87 in Table 26). The A35Rdeletion did not decrease the toxicity of the GLV-1h73 or GLV-1h74strains (see GLV-1j88, 1j89 in Table 26).

TABLE 27 Median tumor volumes at different time points after i.v.injection of different virus strains into nude mice bearing GI-101Atumors Days post- implantation of Median tumor volume (mm³) tumor GLV-GLV- GLV- GLV- GLV- GLV- cells No Treatment 1h68 1h73 1h74 1j87 1j881j89 34 305.2 306.6 351.1 534.75 377.5 353.3 325.45 41 459.2 444.75590.65 881.5 673.35 677.45 753.05 49 794.85 777.05 923.15 1259.8 1126.85988 1095.95 57 1253.4 1102.95 1109.5 1181.4 1271.95 1023.85 975.75 64 *1204.5 691.85 280.5 1125.85 715.45 497.5 71 * 1239.85 318.15 33.81130.35 340.9 * 78 * 1347.2 106.65 * 931.9 * * 85 * 1261.95 8.05 *755.2 * * 92 * 1061.15 0 * 599.5 * *

B. Effects of A35R Deletion on Virulence Following IntranasalAdministration

The in vivo effect of removal of the A35R gene on virulence ofintranasally administered modified vaccinia strains was evaluated.Strains GLV-1h68, GLV-1h73 and GLV-1h74 were evaluated with theircorresponding A35R-deleted strains GLV-1j87, GLV-1j88 and GLV-1j89,respectively. Groups of eight male BALB/c 5-week-old mice wereanesthetized and intranasally challenged with varying concentrations ofeach virus, 1×10⁵, 1×10⁶ or 1×10⁷ PFU, in 20 μl 10 mM Tris-HCl (pH 9.0)or PBS control. Individual mice were weighed three times every week.

Over the observation period, all mice exhibited significant weight gain.The increasing concentrations of each virus slightly decreased thepercentage weight gain; however, the A35R mutation did not significantlyalter the percentage weight gain of corresponding vaccinia viruses inthe mice. Thus, removal of A35R from the viruses does not appear toaffect virulence via intranasal administration.

Example 18 Effects of IL-6 and IL-24 Expressing Viruses on Breast TumorGrowth In Vivo

A. Effects of Modified Viruses on s.c. Human Breast Tumor Xenografts

The in vivo effects of IL-6 expressing viruses GLV-1h90 and GLV-1h91 andGLV-1h92 and IL-24-expressing viruses GLV-1h96, GLV-1h97 and GLV-1h98compared to virus strains GLV-1h68, GLV-1h71 and Dark8.1 on tumor growthwere evaluated using a mouse model of breast cancer. The Dark8.1 strainwas isolated from a culture of GLV-1h68 by selection dark plaques underfluorescence microscope and subsequent plaque purification. Dark8.1 hasan intact F14.5L gene, which is identical in sequence to F14.5L of LIVP.(The lacZ and gusA genes at the TK and HA loci, respectively, in Dark8.1are still intact).

Tumors were established in nude mice by subcutaneously injecting GI-101Ahuman breast carcinoma cells (s.c. on the right lateral thigh; 5×10⁶cells; GI-101A cells: Rumbaugh-Goodwin Institute for Cancer ResearchInc. Plantation, Fla.; U.S. Pat. No. 5,693,533) into female nude mice(Hsd:Athymic Nude-Foxn1^(nu); Harlan, Indianapolis, Ind.) (n=4-8).Thirty three days following tumor cell implantation, groups of mice wereinjected intravenously [in 1001 of PBS, through femoral vein underanesthesia] with 5×10⁶ PFU of GLV-1h68, Dark 8.1, GLV-1h71, GLV-1h90,GLV-1h91, GLV-1h92, GLV-1h96, GLV-1h97 and GLV-1h98, respectively. Thecontrol group of mice was not given any treatment. Tumor volume (mm³)was measured at 31, 39, 54, 62, 69, 76 and 97 days post-cancer cellinjection. Results of median tumor volume are provided in Table 28.GLV-1h90 (expressing IL-6) and GLV-1h96 (expressing IL-24) showedenhanced antitumor response as compared to the corresponding virusGLV-1h68, which does not express IL-6 or IL-24.

TABLE 28 Median tumor volumes at different time points after i.v.injection of different virus strains into nude mice bearing GI-101Atumors Days post- implantation of Median tumor volume (mm³) tumor NoGLV- GLV- GLV- GLV- GLV- GLV- GLV- GLV- cells Treatment 1h68 Dark 8.11h71 1h90 1h91 1h92 1h96 1h97 1h98 31 777.55 578 523.7 571.4 445.6 485.6609 505.1 505.3 716.4 39 1124.4 967.8 1147.6 1012.95 942 860 905.4 939.4829.8 1326.3 54 3535.1 2059.4 1997 1991 1110.8 521.8 811.7 1864.3 1456.92266.8 62 * 2669.05 1184.3 2144.8 1337.3 * * 1969.9 2326.3 2193.2 69 *578 1008.25 * 1219.4 * * 1820.75 2483.55 1957.8 76 * * 452.15 *1378.9 * * 1488.85 2243.25 1419.6 97 * * 154.5 * 155.4 * * 311.3 1131.5945.1

Example 19 Effects of Modified Viruses on Pancreatic Tumor Growth InVivo A. Effects of Modified Viruses on Human Pancreatic Tumors—PANC-1Model

The in vivo effects of GLV-1h68, GLV-1h71, GLV-1h73, GLV-1h81(hk5-expressing), GLV-1h90 (sIL-6R-IL-6 expressing) and GLV-1h96 (IL-24expressing) viruses were evaluated using a mouse model of humanpancreatic cancer. Tumors were established in nude mice bysubcutaneously injecting 5×10⁶ PANC-1 human pancreatic carcinoma cells(ATCC No. CRL-1469) subcutaneously in right lateral thigh of male nudemice (Hsd:Athymic Nude-Foxn1^(nu); Harlan, Indianapolis, Ind.; n 3-8mice/group). Twenty seven days following tumor cell implantation, groupsof mice were injected intravenously [in 100 μl of PBS, through femoralvein under anesthesia] with 5×10⁶ PFU of GLV-1h68, GLV-1h71, GLV-1h73,GLV-1h81, GLV-1h90 and GLV-1h96, respectively. The control group of micewas not given any treatment. Tumor volume (mm³) was measured at 26, 33,41, 49, 56, 68, 76 and 85 days post-cancer cell injection. Results ofmedian tumor volume are provided in Table 29.

GLV-1h81 (hk5-expressing), GLV-1h90 (sIL-6R-IL-6 expressing), andGLV-1h96 (IL-24 expressing) viruses showed significantly acceleratedantitumor responses as compared to GLV-1h68 (Table 29). Among these fourviruses, GLV-1h96 showed the best antitumor activity. In addition, basedon net body weight changes, mice treated with GLV-1h81, GLV-1h90, andGLV-1h96 gained 5-10% more weight than mice treated GLV-1h68, which mayindicate that GLV-1h81, GLV-1h90, and GLV-1 h96 are less toxic to mice(Table 30). Nonetheless, mice treated with GLV-1h68, GLV-1h81, GLV-1h90,and GLV-1h96 all gained significant weight during the course of viraltreatment.

TABLE 29 Median tumor volumes at different time points after i.v.injection of different virus strains into nude mice bearing PANC-1tumors Days post- implantation of Median tumor volume (mm³) tumor GLV-GLV- GLV- GLV- GLV- GLV- cells No Treatment 1h68 1h71 1h73 1h81 1h901h96 26 234 252.2 195.25 171.6 197.75 191.4 169.05 33 387.8 458.25 405.5352.15 352.8 492.9 384.65 41 669 796.25 625.25 638.15 547.1 633.15 547.149 834.7 877.7 480.4 552.45 645.55 720.65 341.25 56 1258.8 823.35 384.95313 589.2 555 229.5 68 1990 616.35 303.35 291.25 422.6 262 157.2 763056.1 436.95 236.6 * 362.55 182.95 124.4 85 4627.4 307.25 201.1 *256.85 133.1 81.15 98 * 218.4 119.7 * 172.45 129.55 41.8 106 *157.95 * * 141.25 110.7 43.8

Comparison of tumor volumes for each of the eight individual miceinjected with either GLV-1h68 (Mice ID Nos. 6060-6067) or GLV-1h90 (MiceID Nos. 6116-6123) are presented in Tables 30 and 31, respectively.Variations in tumor sizes were seen at different time points in the micefollowing GLV-1h90 injection; however, by 80 days following treatment,only tumor remnants remained in most mice. Much smaller variations intumor sizes were seen at different time points in mice treated withGLV-1h68, though the average tumor response was significantly slower ascompared to GLV-1h90. Similar to mice treated with GLV-1h90, only tumorremnants were seen by 80 days after GLV-1h68 injection.

TABLE 30 Median tumor volumes at different time points after i.v.injection of GLV-1h68 into nude mice bearing PANC-1 tumors Days post-implan- tation of tumor Median tumor volume (mm³) cells 6060 6061 60626063 6064 6065 6066 6067 26 272.1 193.3 257.8 339.4 163.1 165.2 246.6351.2 33 474.6 539.1 426.8 578.1 294.7 340.6 441.9 649.6 41 819.5 997.9731.9 1096.9 460.5 532.6 773.9 818.6 49 981.5 815.3 911.8 930.7 557.9843.6 764.3 1153.2 56 819.9 762.7 826.8 757.5 857.5 956.2 553.6 1064.968 812 601.2 484.9 631.5 715.8 445.7 338.8 703.2 76 564.9 593.9 332.4374.8 634.3 228 284.3 499.1 85 488.6 528.7 256.7 311.3 635.3 137.5 301.6303.2 98 301.9 402.8 218.4 123.4 507.3 64.2 187.4 nd 106 210.8 176.7128.5 nd 259.9 45.6 139.2 nd

TABLE 31 Median tumor volumes at different time points after i.v.injection of GLV-1h90 into nude mice bearing PANC-1 tumors Days post-implantation of tumor Median tumor volume (mm³) cells 6116 6117 61186119 6120 6121 6122 6123 26 377 169.9 209.3 281.8 173.5 137.3 130.8212.7 33 631.2 532 453.8 785.6 323.5 370.3 291.9 749.3 41 850.3 757.8497.7 881 508.5 416.5 294.5 958.4 49 755.9 691.5 576 935.4 749.8 360.4205.2 1178.8 56 668.3 589.4 352.5 520.6 765.4 161.7 109.4 879.9 68 345.4332.4 152.9 227.2 748.5 75.8 84.5 296.8 76 233.2 298.3 98.6 219.2 598.7102.3 69.2 146.7 85 164.8 136.1 74.7 130.1 610.2 43.8 64.6 168.1 98148.8 167.3 51.2 110.3 587.9 63.4 0 211.6 106 137.5 125.6 41.7 95.8282.9 68.6 0 269.1

B. Effects of Modified Viruses on Human Pancreatic Tumors—MIA PaCa-2Model

The in vivo effects of GLV-1h68, GLV-1h72, GLV-1h73, GLV-1h81, GLV-1h90and GLV-1h96 were evaluated using a second mouse model of humanpancreatic cancer. Tumors were established in nude mice bysubcutaneously injecting 5×10⁶ cells MIA PaCa-2 human pancreaticcarcinoma cells (ATCC No. CRL-1420) subcutaneously on right lateralthigh of male nude mice (Hsd:Athymic Nude-Foxn1^(nu); Harlan,Indianapolis, Ind.; n=3-8 mice/group). Twenty-nine days following tumorcell implantation, groups of mice were injected intravenously [in 100 μlof PBS, through femoral vein under anesthesia] with 5×10⁶ PFU ofGLV-1h68, GLV-1h72, GLV-1h73, GLV-1h81, GLV-1h90 and GLV-1h96,respectively. The control group of mice was not given any treatment.Tumor volume (mm³) was measured at 30, 36, 45, 52 and 58 dayspost-cancer cell injection. Results of median tumor volume (mm³) areprovided in Table 32. GLV-1h90 (sIL-6R-IL-6 expressing) and GLV-1h96(IL-24 expressing) showed significantly accelerated antitumor responseas compared to GLV-1h68. Among these three viruses, GLV-1h96 showed thebest antitumor activity at 28 days after virus injection.

TABLE 32 Median tumor volumes at different time points after i.v.injection of different virus strains into nude mice bearing MIA-PaCa-2tumors Days post- Median tumor volume (mm³) implantation No GLV- GLV-GLV- GLV- GLV- GLV- of tumor cells Treatment 1h68 1h72 1h73 1h81 1h901h96 30 904.8 761.1 723.7 587 657.6 625.55 527.4 36 1806.4 1482.151457.6 1270.7 1322.7 1067.85 1243.05 45 4641.7 1223.1 1112.3 1180.151508.5 1233.8 1154.3 52 * 1175.85 584.6 749.25 1042.7 853.55 736.45 58 *1073.15 467.8 603.2 984.8 681.95 546.05

C. Effects of Viruses on Body Weight in a Mouse Model of HumanPancreatic Tumors—PANC-1 Model

The percentage of body weight change following intravenousadministration of the viruses in the PANC-1 mouse model of humanpancreatic cancer was also examined (Table 33). Percentage of bodyweight change was measured for the experiment described in Section A.Mice treated with GLV-1h68, GLV-1h81 and GLV-1h90 gained significantweight (all much better than the untreated group) during the course ofviral treatment.

TABLE 33 Body weight change at different time points after i.v.injection of different virus strains into nude mice bearing PANC-1tumors Days post- Body weight Change (%) implantation No GLV- GLV- GLV-GLV- GLV- GLV- of tumor cells Treatment 1h68 1h71 1h73 1h81 1h90 1h96 260 0 0 0 0 0 0 33 −0.36 −2.4 −0.59 −4.76 −3.72 −6.06 −5.09 41 0 −6.85−2.74 −4.17 −5.02 −7.39 −4.28 49 −2.91 −2.78 11.74 7.14 3.35 3.98 11.8156 1.09 7.04 9.98 9.52 11.15 16.29 16.5 68 −3.27 11.11 12.33 −13.4915.24 17.05 11.2 76 −3.27 11.3 8.61 nd 15.8 21.4 10.59 85 −6.18 13.75.68 nd 18.22 22.16 −0.41

Example 20 IL-6 ELISA Correlation of IL-6 with Tumor Volume

The relationship between tumor volume and the amount of IL-6 expressedby injected viruses was evaluated in a mouse model of pancreatic cancer.Tumors were established in nude mice by subcutaneously injecting 5×10⁶cells PANC-1 human pancreatic carcinoma cells (ATCC No. CRL-1469)subcutaneously on right lateral thigh of male nude mice (Hsd:AthymicNude-Foxn1^(nu); Harlan, Indianapolis, Ind.; n=8 mice/group).Twenty-seven days following tumor cell implantation, groups of mice wereinjected intravenously [in 100 μl of PBS, through femoral vein underanesthesia] with 5×10⁶ PFU of either GLV-1h68 or GLV-1h90. At 56 dayspost-cancer cell injection, tumor volume (mm³) was measured and samplesof tumor fluid (˜10-20 μl through needle puncture) and blood serum (˜500μl through retroorbital bleeding) were collected to measure IL-6concentration by ELISA.

For comparison, production of IL-6 was also measured from CV-1 cellsinfected with virus. CV-1 cells in 6-well plates (1.0×10⁶ cells/well)were mock infected or infected in triplicate with GLV-1h68, -1h90, -1h91or -1h92 at m.o.i of 10 for 1 hour at 37° C. The inoculum was aspiratedand the cell monolayers were washed twice with 2 ml of DPBS (Mediatech,Inc., Herndon, Va.). Two ml of DMEM-2 were added into each well. Theinfected medium was collected at 24 h post infection and clarified bycentrifugation at 3,000 rpm for 5 min.

The concentration of human IL-6 in the culture supernatants, mouse sera,and tumor fluids was quantified by Human IL-6 ELISA kit (Cell Sciences,Inc., Catalog No: CKH106) following the manufacturer's instructions. Theculture supernatant samples were diluted 1:1000 or 1:5000, the mouseserum samples were diluted 1:100, and the tumor fluid samples werediluted 1:300. The standards and test samples were assayed in duplicate,and the absorbance at 450 nm was measured. The average absorbanceobtained for each standard was used to generate the standard curve. Theconcentration of human IL-6 in each test sample was interpolated fromthe standard curve. Results for the IL-6 ELISA are shown in Table 34.

TABLE 34 IL-6 concentration following in vitro infection of CV-1 cellsor in vivo i.v. injection of different virus strains into nude micebearing PANC-1 tumors Concen- OD-OD.bl IL-6 Dilution tration Sample (450nm) (ng/ml) Factor (mean) Cell Mock-1 −0.002 n.d. 1:1000 n.d. CultureMock-2 −0.002 n.d. 1:1000 Supernatant Mock-3 −0.001 n.d. 1:1000GLV-1h68-1 0.001 n.d. 1:1000 n.d. GLV-1h68-2 0.001 n.d. 1:1000GLV-1h68-3 −0.002 n.d. 1:1000 GLV-1h90-1 0.553 0.031 1:1000   42 ng/mlGLV-1h90-2 0.703 0.042 1:1000 GLV-1h90-3 0.806 0.053 1:1000 GLV-1h91-11.159 0.108 1:5000  635 ng/ml GLV-1h91-2 1.410 0.162 1:5000 GLV-1h91-31.171 0.111 1:5000 GLV-1h92-1 1.071 0.092 1:5000  562 ng/ml GLV-1h92-21.232 0.122 1:5000 GLV-1h92-3 1.243 0.123 1:5000 Mouse GLV-1h68 (8)−0.001 n.d. 1:100 n.d Serum GLV-1h90-1 0.0912 0.0055 1:100 0.55 ng/mlGLV-1h90-2 0.0496 0.0021 1:100 0.21 ng/ml GLV-1h90-3 0.0694 0.0034 1:1000.34 ng/ml GLV-1h90-4 0.1250 0.0082 1:100 0.82 ng/ml GLV-1h90-5 0.11960.0075 1:100 0.75 ng/ml GLV-1h90-6 0.0466 0.0020 1:100 0.20 ng/mlGLV-1h90-7 0.0046 0.0014 1:100 0.14 ng/ml GLV-1h90-8 0.1591 0.0089 1:1000.89 ng/ml Tumor GLV-1h68 (8) −0.001 n.d. 1:300 n.d. Fluid GLV-1h90-11.125 0.173 1:300 51.9 ng/ml GLV-1h90-2 1.481 0.277 1:300 83.1 ng/mlGLV-1h90-3 0.377 0.026 1:300  7.8 ng/ml GLV-1h90-4 1.312 0.196 1:30058.8 ng/ml GLV-1h90-5 0.793 0.071 1:300 21.3 ng/ml GLV-1h90-6 0.6720.054 1:300 16.2 ng/ml GLV-1h90-7 0.779 0.069 1:300 20.7 ng/mlGLV-1h90-8 1.143 0.140 1:300 42.0 ng/ml OD.bl = OD value measured forthe blank control. n.d. = not detected.

Results for median tumor volume compared to IL-6 concentration areprovided in Table 35 for GLV-1h90, which expresses IL-6, and Table 36for GLV-1 h68, which does not express IL-6. In the 1h90 treated animals,high amounts of IL-6 were found in the cell culture, tumor fluid andblood serum as compared to the samples from mice treated with 1 h68control, which does not express IL-6.

During the tumor shrinking phase of the treatment (56 days postimplantation of the tumor cells), in the GLV-1h90 treated animals, theconcentration of IL-6 in the tumor fluid and in the serum is positivelycorrelated to the tumor volume. This is because the larger tumor volumesustains higher virus replication. The smaller tumors, which are alreadyshrunk by the virus treatment, have less tumor tissue in which the viruscan replicate, and thus display a lower level of IL-6. In the controlGLV-1h68 treated animals, the animals intrinsically express low levelsof IL-6; however, there is no correlation between tumor volume and IL-6concentration.

TABLE 35 IL-6 concentration versus tumor volume following i.v. injectionof GLV-1h90 into mice bearing PANC-1 tumors Tumor Tumor [IL-6] Mouse ID[IL-6] in volume volume in (GLV-1h90 tumor fluid (mm³) (mm³)/ seruminjected) (1:300) at 56 days 500 fluid 6116 1.1248 668.3 1.34 0.912 61171.4813 589.4 1.18 0.496 6118 0.3774 352.5 0.71 0.694 6119 1.3121 520.61.04 1.25 6120 0.7927 765.4 1.53 1.196 6121 0.6723 161.7 0.32 0.40666122 0.7785 109.4 0.22 0.046 6123 1.1425 879.9 1.76 1.591

TABLE 36 IL-6 concentration versus tumor volume following i.v. injectionof GLV-1h68 into mice bearing PANC-1 tumors Tumor Tumor [IL-6] Mouse ID[IL-6] in volume volume in (GLV-1h68 tumor fluid (mm³) (mm³)/ seruminjected) (1:300) at 56 days 500 fluid 6060 −0.0102 819.9 0.0041 −0.02486061 −0.0077 762.7 0.00381 −0.0108 6062 −0.0032 826.8 0.004134 −0.00886063 0.0072 757.5 0.00379 0.0044 6064 0.0093 857.5 0.00429 0.0017 60650.0087 956.2 0.004781 −0.0089 6066 −0.0049 553.6 0.002768 −0.0157 6067−0.0042 1064.9 0.00532 −0.0093

Example 21 Effects of Modified Viruses on Prostate Tumor Growth In VivoA. Effects of Viruses Administered to Female Nude Mice on Human ProstateCarcinoma

The in vivo effects of GLV-1h68, GLV-1h90, GLV-1h96 or a combination ofGLV-1h90 and GLV-1h96 were evaluated using two mouse models of humanprostate cancer. In the first model, the in vivo effects of GLV-1h68,GLV-1h90, GLV-1h96 or a combination of GLV-1h90 and GLV-1h96 on DU145human prostate tumors was assessed. Tumors were established bysubcutaneous implantation of 1×10⁷ DU145 human prostate cancer cells(ATCC# HTB-81) in the right lateral thigh of male nude mice (Hsd:AthymicNude-Foxn1^(nu); Harlan, Indianapolis, Ind.; n=3-8 mice/group). Nineteendays following tumor cell implantation, groups of mice were injectedintravenously [in 100 μl of PBS, through femoral vein under anesthesia]with 5×10⁶ PFU of GLV-1h68, GLV-1h90, or GLV-1h96, or 2.5×10⁶ PFU eachof GLV-1h90 and GLV-1h96. Median tumor volumes (mm³) were measured usinga digital caliper on day 18, 25, 31, 39, 45, 54, and 61 (days aftertumor cell implantation). Results are shown in Table 37a.

In the second model, the in vivo effects of GLV-1h68 on PC-3 humanprostate tumors was assessed. Tumors were established by subcutaneousimplantation of 1×10⁷ PC-3 human prostate cancer cells (ATCC# CRL-1435)in the right lateral thigh of nude mice (Hsd:Athymic Nude-Foxn1^(nu);Harlan, Indianapolis, Ind.). Following tumor cell implantation, groupsof mice were injected intravenously either through the femoral vein(f.v.) or through the tail vein (t.v.) with 5×10⁶ PFU of GLV-1h68 in 100μl of PBS. Median tumor volumes (mm³) were measured using a digitalcaliper on days 27, 42, 50, 56, 63, 71, 78, 86, 105, 114, 133 and 146after tumor cell implantation. Results are shown in Table 37b.

GLV-1h90 (sIL-6R-IL-6 expressing), GLV-1h96 (IL-24 expressing), andGLV-1h90 plus GLV-1h96 combination treatments of mice bearing DU145tumors showed significantly accelerated and enhanced antitumor responseas compared to GLV-1h68 (Table 37a). Among these four treatment groups,GLV-1h96 and GLV-1h90 plus GLV-1h96 combination treatments showed thebest antitumor activities. Tumors were eradicated in almost all mice inthese two treatment groups 43 days after virus injection.

TABLE 37a Median tumor volumes at different time points after i.v.injection of different virus strains into nude mice bearing DU145 tumorsMedian tumor volume (mm³) GLV- Days post- 1h90 + implantation No GLV-GLV- GLV- GLV- of tumor cells Treatment 1h68 1h90 1h96 1h96 18 423.55430.55 440.6 436.9 388.3 25 794.55 703.75 832.35 803.35 751.05 311036.05 1011.2 1025 952.3 1045.45 39 1278.9 1123.05 828.15 910.4 923.1545 1649.9 1031 639.25 644.75 449.7 54 1746.8 951.5 452.15 238.75 214.0561 2068.8 766.1 369.8 5.15 87.25

Administration of GLV-1h68, via to the tail vein or femoral vein, tomice bearing PC-3 tumors resulted in similar tumor progression to thatseen in mice that were not treated (Table 37b). Administration ofGLV-1h68 via the tail vein resulted in slightly slower tumor growthcompared with tumor growth in untreated mice.

TABLE 37b Median tumor volumes at different time points after i.v.injection into the tail vein or femoral vein of GLV-1h68 into nude micebearing PC-3 tumors Days post- implantation of Median tumor volume (mm³)tumor GLV-1h68 GLV-1h68 cells No Treatment (t.v.) (f.v.) 27 38.05 28.5537.8 42 158.25 145.8 111.45 50 188.15 247.25 146.2 56 215.3 259.45 18763 373.15 330.55 323.5 71 446.1 381.15 322.05 78 546.15 525.9 408.9 86679.8 718.1 549.7 105 1335.35 1000.9 1114.85 114 1499.9 1423 1522.8 1332685.2 2162.4 2719.8 146 3342.45 2627.95 3120.1

B. Effects of Viruses on Body Weight in a Mouse Model of Human ProstateCancer

The percentage of body weight change following intravenousadministration of the viruses in the mouse model of human prostatecancer was also examined (Table 38. Percentage of body weight change wasmeasured for the experiment described in Section A. Mice in alltreatment groups gained significant weight (equal to or better than theuntreated group) and remained healthy during the course of viraltreatment.

TABLE 38 Body weight change at different time points after i.v.injection of different virus strains into nude mice bearing DU145 tumorsBody weight Change (%) GLV-1h90 + No GLV- GLV- GLV- GLV- Days Treatment1h68 1h90 1h96 1h96 18 0 0 0 0 0 25 6.39 1.62 −1.3 0.81 1.46 31 5.010.97 −0.49 2.75 0.49 39 5.01 4.7 6.68 3.23 1.79 45 9.67 6.16 9.78 7.277.97 54 9.15 9.89 7.98 5.33 9.11 61 5 10.5 7.98 10.18 5.53

Example 22 Effect of Erbitux or Avastin Combination Therapies on HumanPancreatic Carcinomas In Vivo

A. Effects of Modified Viruses with Erbitux or Avastin on Tumor Growth

Tumors were established in nude mice by subcutaneously injecting 5×10⁶cells PANC-1 human pancreatic carcinoma cells (ATCC No. CRL-1469)subcutaneously on the right lateral thigh of male nude mice (Hsd:AthymicNude-Foxn1^(nu); Harlan, Indianapolis, Ind.; n=3-8 mice/group). Twentyseven days following tumor cell implantation, groups of mice wereinjected intravenously [in 100 μl of PBS, through femoral vein underanesthesia] with 5×10⁶ PFU of GLV-1h68. For the combination treatments,Eribitux was administered i.p. at a dose of 3 mg/kg twice a week forfive consecutive weeks) and Avastin was administered i.p. at a dose of 5mg/kg twice a week for five consecutive weeks. The control group of micewas not given any treatment. Tumor volume (mm³) was measured at 26, 33,41, 49, 56, 68, 76, 85, 98 and 106 days post-cancer cell injection.Results of median tumor volume are provided in Table 39. Avastinexhibited a significant enhancement tumor regression when used incombination with 1h68, whereas Erbitux treatment slightly improved tumorregression when used in combination with 1h68. In addition, miceremained healthy following 1h68 and Avastin treatment.

TABLE 39 Median tumor volumes at different time points after i.v.injection of GLV-1h68 into nude mice bearing PANC-1 tumors with orwithout Erbitux or Avastin combination therapy Median tumor volume (mm³)GLV- GLV- No GLV- 1h68 + 1h68 + Days Treatment 1h68 Erbitux ErbituxAvastin Avastin 26 234 252.2 170.9 190.15 230.75 221.5 33 387.8 458.25420 382.45 422.05 366.3 41 669 796.25 695.15 507.7 628.7 537.65 49 834.7877.7 724.3 785.65 648.5 742.8 56 1258.8 823.35 617.8 1126.35 449.6988.9 68 1990 616.35 437.1 1926.6 275.55 1450 76 3056.1 436.95 382.92684.15 255.45 1919.85 85 4627.4 307.25 294.3 4283.6 205.3 2556.3 98 *218.4 236.2 * 141 * 106 * 157.95 212.05 * 143.8 *

B. Effects of Combination Therapy on Body Weight in a Mouse Model ofHuman Pancreatic Cancer

The percentage of body weight change following intravenousadministration of the viruses in the PANC-1 mouse model of humanpancreatic cancer was also examined (Table 40). Percentage of bodyweight change was measured for the experiment described in Section A.

Mice injected with GLV-1h68 alone, GLV-1h68 plus Erbitux, and GLV-1h68plus Avastin treatment groups gained significant weight, which wassignificantly better than the untreated, treated with Erbitux alone, ortreated with Avastin alone groups, and remained healthy during thecourse of viral treatment.

TABLE 40 Body weight change at different time points after i.v.injection of GLV-1h68 into nude mice bearing PANC-1 tumors with orwithout Erbitux or Avastin combination therapy Body weight Change (%)Days post- GLV- GLV- implantation No GLV- 1h68 + 1h68 + of tumor cellsTreatment 1h68 Erbitux Erbitux Avastin Avastin 26 0 0 0 0 0 0 33 −0.36−2.4 −3.1 −0.19 −4.41 −1.14 41 0 −6.85 −5.43 0.38 −4.41 −3.6 49 −2.91−2.78 1.55 −2.66 2.61 −2.65 56 1.09 7.04 9.88 −1.52 14.63 −6.06 68 −3.2711.11 14.53 −5.69 19.84 −5.49 76 −3.27 11.3 13.76 −3.04 19.44 −7.95 85−6.18 13.7 17.25 −4.36 19.44 −8.9

Example 23 Effect of Combination Therapy on Pancreatic Tumor Growth InVivo

A. Combination Therapy with IL-6 or IL-24-Expressing Viruses andGemcitabine

Tumors were established in nude mice by subcutaneously injecting 5×10⁶cells MIA PaCa-2 human pancreatic carcinoma cells (ATCC No. CRL-1420)subcutaneously on right lateral thigh of male nude mice (Hsd:AthymicNude-Foxn1^(nu); Harlan, Indianapolis, Ind.; n=3-8 mice/group).Twenty-nine days following tumor cell implantation, groups of mice wereinjected intravenously [in 100 μl of PBS, through femoral vein underanesthesia] with 5×10⁶ PFU of GLV-1h68, GLV-1h90 or GLV-1h96 viruses.For the combination treatment, Gemcitabine was administered i.p. at adose of 50 mg/kg once every three days for five doses. The control groupof mice was not given any treatment. Tumor volume (mm³) was measured at30, 36, 45, 52, and 58 days after tumor cell implantation. Results formedian tumor volume are provided in Table 41. GLV-1h68 plus Gemcitabinecombination treatment showed significantly accelerated and enhancedantitumor response as compared to treatment with GLV-1h68 alone or withGemcitabine alone.

TABLE 41 Median tumor volumes at different time points after i.v.injection of different modified viruses into nude mice bearingMIA-PaCa-2 tumors with or without Gemcitabine combination therapy Mediantumor volume (mm³) Days post- GLV- GLV- GLV- implantation No GLV- GLV-GLV- 1h68 + 1h90 + 1h96 + of tumor cells Treatment 1h68 1h90 1h96Gemcitabine Gemcitabine Gemcitabine Gemcitabine 30 904.8 761.1 625.55527.4 706.7 597.8 633.6 739.2 36 1806.4 1482.15 1067.85 1243.05 1556.21390.8 1209.15 1413.6 45 4641.7 1223.1 1233.8 1154.3 3184.35 12091315.45 1593.4 52 * 1175.85 853.55 736.45 4187.45 854.4 995.5 1049.458 * 1073.15 681.95 546.05 * 766.6 825.65 981.35B. Comparison of Combination Therapy with Gemcitabine or Avastin

Tumors were established in nude mice by subcutaneously injecting 5×10⁶cells MIA PaCa-2 human pancreatic carcinoma cells (ATCC No. CRL-1420)subcutaneously on right lateral thigh of male nude mice (Hsd:AthymicNude-Foxn1^(nu); Harlan, Indianapolis, Ind.; n=3-8 mice/group).Twenty-nine days following tumor cell implantation, groups of mice wereinjected intravenously [in 100 μl of PBS, through femoral vein underanesthesia] with 5×10⁶ PFU of GLV-1h68 viruses. For the combinationtreatment, Avastin was administered i.p. at a dose of 5 mg/kg twice aweek for five consecutive weeks. The control group of mice was not givenany treatment. Tumor volume (mm³) was measured at 30, 36, 45, 52, and 58days after tumor cell implantation. Results for median tumor volume areprovided in Table 42. Combination between 1h68 and Avastin orGemcitabine exhibited a significant synergistic effect as comparedGLV-1h68, Avastin or Gemcitabine alone.

TABLE 42 Median tumor volumes at different time points after i.v.injection of GLV-1h68 into nude mice bearing MIA-PaCa-2 tumors with orwithout Gemcitabine or Avastin combination therapy Median tumor volume(mm³) Days post- GLV- GLV- implantation No GLV- 1h68 + 1h68 + of tumorcells Treatment 1h68 Avastin Avastin Gemcitabine Gemcitabine 30 904.8761.1 645.65 824.5 706.7 597.8 36 1806.4 1482.15 1408.4 1325.6 1556.21390.8 45 4641.7 1223.1 2746.25 1422.45 3184.35 1209 52 * 1175.854241.15 842.05 4187.45 854.4 58 * 1073.15 * 697.9 * 766.6

Example 24 Effect of Irinotecan Combination Therapy on Breast TumorGrowth In Vivo

Tumors were established in nude mice by subcutaneously injecting 5×10⁶cells GI-101A human breast carcinoma cells (Rumbaugh-Goodwin Institutefor Cancer Research Inc. Plantation, Fla.; U.S. Pat. No. 5,693,533)subcutaneously on the right lateral thigh of female nude mice(Hsd:Athymic Nude-Foxn1^(nu); Harlan, Indianapolis, Ind.;n=4-8mice/group). Following tumor cell implantation, one group of micewas injected with 5×10⁶ PFU/mouse of GLV-1h68 virus in the femoral veinat 23 days post-cancer cell injection, one group of mice wasintraperitoneally injected with 80 mg/kg irinotecan once a week on eachof days 36, 43, 50 and 59 post-cancer cell injection, and one groupreceived combination therapy of GLV-1h68 and irinotecan (irinotecaninjected at same time points as the last group). Tumor volume (mm³) wasmeasured at days 22, 32, 36, 42, 49, 59, 66, 80, 86, 99, 108, and 116post-cancer cell injection. Results are provided in Table 43.Combination between 1 h68 and irinotecan exhibited a significantsynergistic effect as compared GLV-1h68 or irinotecan alone.

TABLE 43 Median tumor volumes at different time points after i.v.injection of GLV-1h68 into nude mice bearing GI-101A tumors with orwithout Irinotecan combination therapy Days post- implantation Mediantumor volume (mm³) of GLV- tumor No GLV- 1h68 + cells Treatment 1h68Irinotecan Irinotecan 22 244.5 166 184 219.45 32 594.9 604.3 458.45 54636 695.85 649.3 546.2 734.55 42 951.55 1002 668.75 921.25 49 1261.051200.8 712.9 973.35 59 1844.7 1694 831.8 901.55 66 nd 2049.2 1011.81003.25 80 nd 2608.4 1502.15 817.45 86 nd 2296.8 1678.4 684.8 99 nd1647.5 2712.55 526.15 108 nd nd nd 429.25 116 nd nd nd 337.65

Example 25 Expression of Anti-VEGF Single Chain Antibody by ModifiedVaccinia Strains A. Expression of G6-FLAG in CV-1 Cells

Monkey CV-1 cells were infected with the GLV-1h107, GLV-1h108 andGLV-1h109 virus strains and expression of G6-FLAG was investigated viaWestern blot analysis. For negative controls, uninfected cells or cellsinfected with GLV-1h68, which lacks the G6-FLAG gene, were used. At 48 hpost-infection, the supernatant of the infected cells was collected andcell pellets were harvested. The supernatant samples were centrifuged toremove cellular debris. Protein fractions were denatured and separatedvia vertical SDS-PAGE (sodium-dodecyl-sulfate polyacrylamide gelelectrophoresis). Proteins were transferred to a PVDF-membrane andnonspecific binding was blocked by incubation of the membrane in1×PBS/5% skim milk. The membrane was then incubated with the specificantibody rabbit anti-DDDDK-tag (detects the FLAG-tag) overnight.Chromogenic detection was achieved using a secondary HRP-conjugated goatanti-rabbit-IgG and an HRP detection kit, Opti4CN (4-chloro-1-naphthol;Bio-Rad). Expressed scAb G6-FLAG protein (approximate size 32 kDa) wasdetected both in the supernatant and the pellet (intracellular protein)for all three strains. Both GLV-1h108 (P_(SEL)) and GLV1h109 (P_(SL))strains exhibit high levels of expression of G6-FLAG after 48 hpost-infection due to the stronger P_(SEL) and P_(SL) promoters in the1h108 and 1h109 strains, respectively. Strain GLV-1h107 (P_(SE))exhibits a lower expression of G6-FLAG as compared to GLV-1h108 andGLV-1h109 due the weaker P_(SE) promoter in the GLV-1h107 strain. Theexperiment shows that the recombinant DNA can be delivered to mammaliancells via viral delivery by the modified vaccinia strains. Therecombinant protein is successfully expressed in the infected cell andis secreted into the surrounding medium.

During the course of virus replication, infected cells undergo apoptosisand die. The cell membrane is also destroyed and cellular proteins arereleased into the supernatant. In order to demonstrate that the proteinin the collected supernatants was a result of secretion by the infectedcells and not by release of proteins into the supernatant by dying ordead cells, supernatant was harvested from infected CV-1 cells at 6 hpost-infection and analyzed by Western blot detection. Since cell deathis minimal at earlier time points during infection, analyzing thesupernatant from the infected cells at 6 h post-infection allowsdetection of proteins that are actively secreted into the supernatant.By Western blot analysis, expression at 6 h post-infection was low butdetectable in the cell supernatants, indicating that the G6-FLAG fusionprotein is expressed and secreted by the cells into the surroundingmedia

B. Expression of G6-FLAG in Tumor Cell Lines

Different tumor cell lines were infected with an MOI of 10 (GLV-1h107 toGLV-1h109; GLV-1h68 as a control). The used cell lines included breasttumor (GI-101A), prostate adenocarcinoma (PC-3), colon carcinoma (HT-29)and pancreatic cancer (PANC-1) cell lines. After 48 h post-infection,the supernatant of the infected cells was collected and cell pelletswere harvested. The supernatant samples were centrifuged to removecellular debris. Western blot analysis was performed as described in(A). The expressed scAb G6-FLAG (approximate size 32 kDa) was detectedafter 48 h for all strains tested. Both GLV-1h108 (P_(SEL)) and GLV1h109(P_(SL)) strains exhibited higher levels of expression of G6-FLAG ascompared to strain GLV-1h107 (P_(SE)). The supernatant protein fractionscontain a higher amount of protein compared to pellet protein fractions.

Example 26 Functional In Vitro Analysis of Expressed G6-FLAG by ELISA

In order to investigate the functional properties of the scAb G6-FLAG,CV-1 cells were infected with virus strains GLV-1h107, GLV-1h108 andGLV-1h109, and the binding of expressed G6-FLAG, collected from cellsupernatants, to human VEGF protein was analyzed by Enzyme-LinkedImmunoSorbent Assay (ELISA). Uninfected CV-1 cells and CV-1 cellsinfected with GLV-1h68 were employed as negative controls. Microtiterplates were pre-incubated with human VEGF (Sigma) at a concentration of1.8 μg/ml at 4° C. overnight. Supernatant of CV-1 infected cells wassampled after 48 h and centrifuged to remove cellular debris. Thesupernatant was then serially diluted and incubated at room temperatureon the pre-coated microtiter plates. To detect functional binding,rabbit anti-DDDDK-tag and HRP-conjugated goat anti-rabbit-IgG antibodieswere serially used. Chromogenic detection was achieved by using TMB(3,3′,5,5′Tetramethylbenzidine, Sigma), and the reaction was stoppedwith 2N hydrochloride acid. The blue color development was measuredusing a microtiter plate reader (Molecular Devices).

The supernatants of cells infected with the GLV-1h107, GLV-1h108, andGLV-1h109 viruses contained functional G6-FLAG protein that bound to theVEGF-coated plates (Table 44). The concentration of G6-FLAG in thesupernatants of the GLV-1h108 or GLV-1h109 infected cells was higher ascompared to the GLV-1h107 infected cells. In the GLV-1h108 or GLV-1h109samples, the supernatants needed to be diluted at least by a factor of50 to achieve unsaturated detection. The controls of uninfected andGLV-1h68 infected cells show marginal or no binding affinity to thehuman VEGF-coated plate.

TABLE 44 Analysis of functional binding of scAb G6-FLAG to human VEGFvia ELISA Absorbance OD₄₅₀ Dilution GLV1h107 GLV1h108 GLV1h109Uninfected CV-1 GLV1h68 1:200 0.0908 0.7637 0.8728 0.2330 0.1033 1:1000.1363 1.0060 1.0220 0.0737 −0.0534 1:50 0.3209 1.3736 1.2368 −0.0498−0.0097 1:20 0.5558 1.9924 1.8959 −0.0006 0.0119 1:10 0.9059 1.61351.8163 0.00607 0.0124 1:5 1.4289 2.0801 1.8982 −0.0012 0.0295 1:2 1.87652.0439 1.9194 0.00807 0.1445

Example 27 Functional In Vitro Analysis of Anti-Angiogenic Activity

The anti-angiogenic effects of virally expressed scAb G6-FLAG can bestudied in an in vitro model of angiogenesis. The murine endothelialcell line 2h11 (ATCC No. CRL-2163) can be employed in order to studyinhibitory effects of the scAb G6-FLAG on tube formation in vitro. Forthe tube formation assays, the cells are trypsinized, counted anddiluted to a concentration of about 1×10⁵ cells/ml. Human VEGF (Sigma)is added to the cells (end concentration 40 ng/ml) and mixed. CV-1 cellsare separately infected with GLV-1h107, GLV-1h108 or GLV-1h109 (scAbG6-FLAG-expressing viruses) or control strain GLV-1h68. The supernatantof infected CV-1 cells is harvested and centrifuged in order to removecellular debris. Several different volumes of the CV-1 cell supernatantare then added to the samples of 2h11 endothelial cells. After a 5-10min incubation, the suspension is added to the wells of a microtiterplate (24-well; 500 μl/well) containing a layer of Matrigel™ (BDBiosciences; other matrices can also be employed to induce tubeformation, for example, fibrin gels or gelatin matrices). Tube formationis monitored with a microscope over time (approx. 24 h). The followingcontrols can be used: 1) cells incubated without addition of VEGF(negative control); 2) cells incubated with VEGF alone; or 3) cellsincubated with VEGF and different concentrations of Avastin® (positivecontrol). The latter control mimics the mode of action of the scAbG6-FLAG by binding VEGF and sequestering it away from its receptor onthe endothelial cells.

Example 28 Purification of G6-FLAG from Supernatants of Virus InfectedCells

The virally expressed G6-FLAG protein can be purified for furtheranalytical studies in order to reduce background activity of otherfactors present in the supernatant samples collected from virus infectedcells. The scAb G6-FLAG protein was immunoprecipitated via the FLAG-tagusing Sigma FLAG®-Tagged Protein Immunoprecipitation Kit according tothe manufacturer's recommendations. FLAG-tagged proteins were bound byan antibody, which is bound to an agarose resin. Following binding andwashing off unbound supernatant proteins, the G6-FLAG protein was elutedfrom the resin by competitive binding with a short FLAG peptide. Theisolated G6-FLAG can be kept in a native condition followingpurification.

The purified proteins were analyzed by Western blot detection and ELISAassays. G6-FLAG protein (32 kDa) was detected in the GLV-1h107,GLV-1h108 and GLV-1h109 samples. FLAG-BAP Fusion Protein (55 kDa; Sigma)was employed as a positive control for the protein detection. No G6-FLAGprotein was present in supernatant from control GLV-1h68 infected cellsor in the PBS control. The functionality of the purified G6-FLAG proteinwas analysed by ELISA assay (Table 45). The G6-FLAG protein isolatedfrom the GLV-1h107, GLV-1h108 and GLV-1h109 samples all exhibitedfunctional binding of human VEGF. The sample derived from GLV-1h108 andGLV-1h109 infected CV-1 cells exhibited a higher absorbance as comparedGLV-1h107 infected CV-1 cells due to higher amounts of protein in thestarting sample. This Western blot analysis showed a similar relativeamounts of G6-FLAG in the GLV-1h108 and GLV-1h109 versus GLV-1h107derived samples.

TABLE 45 Analysis of functional binding of purified scAb G6-FLAG tohuman VEGF via ELISA Absorbance OD₄₅₀ Dilution GLV1h107 GLV1h108GLV1h109 GLV1h68 1:10 .2644 1.946 1.1976 −0.0106 1:50 0.0574 0.88531.0877 −0.0093 1:100 0.0167 0.6854 0.8257 0.0185 1:200 0.0111 0.40840.5142 0.006 1:500 0.0021 0.1911 0.3093 0.0067

Example 29 Effects of scAb Anti-VEGF Expressing Viruses on Breast TumorGrowth In Vivo

The in vivo effects of virally expressed G6-FLAG protein on tumor growthwere evaluated in a mouse model of breast cancer. The human breastcancer cell line GI-101A was used as a xenograft tumor model in nudemice. The in vivo effects of G6-FLAG expressing viruses GLV-1h107,GLV-1h108 and GLV-1h109 on tumor growth were compared to virus strainsGLV-1h68 and GLV-1h72.

Tumors were established in nude mice by subcutaneously injecting GI-101Ahuman breast carcinoma cells (s.c. on the right lateral thigh; 5×10⁶cells; GI-101A cells: Rumbaugh-Goodwin Institute for Cancer ResearchInc. Plantation, Fla.; U.S. Pat. No. 5,693,533) into four- tofive-week-old female nude mice (Hsd: Athymic Nude-Foxn1^(nu); Harlan,Indianapolis, Ind.) (n=4-5 per group). Twenty-three days following tumorcell implantation (approximate tumor volume 250 mm³), the groups of micewere injected intravenously [in 100 μl of PBS, through femoral veinunder anesthesia] with 5×10⁶ PFU of GLV-1h68, GLV-1h72, GLV-1h107,GLV-1h108 and GLV-1h109. Tumor dimensions (mm) were measured using adigital caliper, and tumor volume (mm³) was calculated according to theformula: (height-5)×width×length)/2. The net body weight change overtime was determined by weighing mice and subtracting the estimated tumorweight (1000 mm³=1 g) (Table 46). Net body weight change was calculatedby the following formula: [(Gross weight−tumor weight)−(gross weight atT23−tumor weight at T23)]/(gross weight at T23−tumor weight at T23);“T23” means 23 days after tumor cell implantation, which was also thetime of virus injection. The survival rate of mice treated with thedifferent virus strains was monitored throughout the experiment.

Table 46 shows the median GI101A tumor volume over time after virusinjection. In the untreated control mice, exponential tumor growth wasobserved. Mice were killed after tumor growth exceeded 2500 mm³ (58 daysafter tumor implantation). In the GLV-1h68 treated mice, the tumorgrowth shows the typical three-phase growth pattern (Zhang et al. (2007)Cancer Research 67:20). During Phase I (growth phase), the tumor volumeexceeds that of the untreated control group. The tumor growth then slowsand arrests approximately 30 days following virus injection (inhibitoryphase, Phase II). After an additional 10 days the inhibitory phase isfollowed by the regression phase (Phase III) where the tumor volumedecreases over time until the termination of the experiment at 100 daysfollowing tumor cell implantation. Mice infected with the virus strainGLV-1h72 show a different tumor growth pattern than GLV-1h68, which ischaracterized by a delayed onset of tumor growth with no apparent PhaseI and a lower median tumor size in Phase II. The tumor regression rate(in Phase III) in GLV-1h72 treated mice is slower than in GLV-1 h68treated mice. The GLV-1h72 virus strain also is slightly more toxic invivo than GLV-1h68 based on net body weight changes in nude mice aftervirus treatment (Table 47). Tumors in mice treated with the GLV-1h107virus strain show a growth pattern similar to GLV-1h72 treated tumorswith a delayed onset of tumor growth of a week compared to GLV-1h68 andan overall lower tumor size than in the GLV-1h68 treated group (Table46). Forty-two days after virus injection, the regression phase startswith a rapid decrease of tumor volumes. After a week the tumor volumesreach a plateau. Due to the lower survival rate in this group, thenumber of mice fell below the statistically evaluable number. All micein the group were killed one week before the endpoint of the experiment.Further outcome of tumor growth could not be monitored throughout thisexperiment. Toxicity studies as determined by net body weight changeindicate that this virus strain can be more toxic than GLV-1h68, sincethe mice do not show significant weight gain over the course of theexperiment (Table 47).

In mice treated with GLV-1h108, a more pronounced inhibition of tumorgrowth can be seen compared to GLV-1h68. Tumor growth was inhibited veryearly after injection and does not reach comparable tumor sizes as inGLV-1h68 treated mice in the inhibitory phase. Over the course oftherapy, tumor sizes in GLV-1h108 treated mice are lower than inGLV-1h72 treated mice. The regression phase is characterized by a slowertumor volume decrease than in GLV-1h68 treated animals. At the endpointof the experiment, median tumor volumes of the mice are similar to thoseof GLV-1h68 and GLV-1h72 treated mice.

The infection of tumor bearing mice with GLV-1h109 leads to a slowertumor growth as described for GLV-1h108 treated tumors, but the tumorgrowth pattern does not resemble the above described three phasepattern. In contrast, the growth of the tumors in these mice can bedivided into regression and progression phases of tumor growth. After athird regression phase, tumors start to grow again until the terminationof the experiment.

Based on net body weight change data, both GLV-1h108 and GLV-1h109 viralstrains show a slightly increased toxicity as compared to GLV-1h68. Thiscorrelates with a survival rate of 80% in GLV-1h108 and GLV-1h109treated mice, as compared to a survival rate of 100% in GLV-1h68 treatedanimals.

The results of the tumor therapy experiment show that treatment of nudemice bearing GI-101A tumors with the GLV-1h107, GLV-1h108 and GLV-1h109virus strains all lead to a marked regression or delay of tumor growthin vivo. The varying ability of the GLV-1h107, GLV-1h108 and GLV-1h109virus strains to mediate tumor growth inhibition in vivo correlates withthe strength of the different promoters used to express the G6-FLAGgene. GLV-1h107 comprises the weakest promoter for the G6-FLAGexpression, whereas GLV-1h108 and GLV-1h109 both contain the strongerlate promoters and show increased expression of G6-FLAG in vitro.

TABLE 46 Median tumor volumes at different time points after i.v.injection of G6-FLAG-expressing viruses into nude mice bearing GI-101Atumors Median tumor volume (mm3) Days post- No implantation treatmentGLV GLV GLV GLV GLV of tumor cells (PBS) 1h68 1h72 1h107 1h107 1h109 21241.46 2121.975 225.51 218.655 1183.955 984.175 23 294.75 277.49 257.155265.88 238.365 284.605 27 367.385 384.88 317.36 349.455 383.8 358.3 30427.9 573.31 433.37 430.605 424.335 444.54 34 729.68 946.35 628.875627.96 616.6 599.775 41 965.51 1098.17 1020.31 959.9 721.145 887.13 43934.735 1229.04 981.53 985.135 767.175 678.125 48 1167.225 1416.891027.41 1109.095 925.975 739.355 51 1502.02 1664.53 1101.01 1032.565911.125 793.465 55 2121.975 1652.39 1078.65 1183.955 984.175 1006.535 582578.15 1564.61 1004.88 1162.59 826.3 805.38 65 1353.9 1086.465 1094.915795.92 683.36 72 1328.66 1043.175 747.63 745.995 968.455 79 1003.35956.08 796.26 661.61 734.575 87 851.52 970.815 748.61 639.15 900.045 93695.89 634.21 796.23 627.525 1108.115 100 452.82 464.01 436.38 1108.085

TABLE 47 Net animal body weight change (%) during the therapy of GI-101Atumors with G6-FLAG-expressing virus strains Mean weight change in %Days post- No implantation treatment GLV GLV GLV GLV GLV of tumor cells(PBS) 1h68 1h72 1h107 1h107 1h109 0 0 0 0 0 0 0 4 4.7 3.8 4.2 4.3 1.6 17 6.3 1.3 6 5.7 0.9 2.7 11 6.8 3.7 4.8 4.4 −0.5 3.7 18 3 6 7.1 1.9 0.70.2 21 8.3 5.9 12.3 4.8 −0.1 −3.9 25 13 12.2 13.1 7.4 4.1 4.9 28 12 14.69.3 6.1 1.7 6.4 32 8.2 13.8 14.6 6.2 5.1 6.1 35 11.1 15.5 12.2 5.5 8.411 38 13 14.1 13 8.2 9.6 11.5 42 16.1 6.8 4.8 11.3 11.4 49 16.3 12.6 510 9.4 56 18.8 14.1 2.6 12.1 10.4 63 20.6 4.8 9.3 15.7 13.9 70 22.5 15.52.3 16.3 13.8 77 23.3 10.5 14.8 10.9

The survival rate of mice treated with the different viruses wasmonitored for 100 days after the implantation of the tumor cells (77days after injection of the viruses). All of the mice that were treatedwith GLV1h68 survived throughout the monitoring period. All off the micetreated with GLV1h107 survived until day 60 post implantation, at whichpoint one mouse died. Another mouse in this group died at day 80,reducing the final survival rate to 60%. Of the mice that receivedGLV1h108, only one died (day 80 post implantation) resulting in asurvival rate of 80%. An 80% survival rate also was observed in thegroup of mice that were treated with GLV1h109, with one mouse dying 50days post transplantation.

Example 30 Effects of scAb Anti-VEGF Expressing Viruses on PancreaticTumor Growth In Vivo

The in vivo effects of virally expressed G6-FLAG protein on tumor growthwas evaluated in a mouse model of human pancreatic cancer. Tumors wereestablished in nude mice by subcutaneously injecting 5×10⁶ cells MIAPaCa-2 human pancreatic carcinoma cells (ATCC No. CRL-1420)subcutaneously on right lateral thigh of male nude mice (Hsd:AthymicNude-Foxn1^(nu); Harlan, Indianapolis, Ind.; n=3-8 mice/group).Twenty-nine days following tumor cell implantation, groups of mice wereinjected intravenously [in 100 μl of PBS, through femoral vein underanesthesia] with 5×10⁶ PFU of GLV-1h68, GLV-1h107 and GLV-1h109,respectively. The control group of mice was not given any treatment.Tumor volume (mm³) was measured at 30, 36, 45, 52 and 58 dayspost-cancer cell injection. Results of median tumor volume (mm³) areprovided in Table 48.

GLV-1h109 exhibited the best antitumor efficacy, resulting in tumorsthat were 27% the volume of those seen in mice treated with GLV-1h68.GLV-1h109 also exhibited good antitumor activity, reducing the growth oftumors to half that seen in mice treated with GLV-1h68.

TABLE 48 Median tumor volumes at different time points after i.v.injection of different virus strains into nude mice bearing MIA-PaCa-2tumors Days post- implantation Median tumor volume (mm³) of tumor GLV-GLV- GLV- cells No Treatment 1h68 1h107 1h109 30 904.8 761.1 711.7 565.636 1806.4 1482.15 1410.2 1170 45 4641.7 1223.1 1453.9 832 52 * 1175.851098.7 611.7 58 * 1073.15 927.7 568 69 * 942.55 709.25 487.9 80 * 1200.1721.8 455.3 92 * 1720.15 850.45 471.6

Example 31 Effects of IL-6 and IL-24 Expressing Viruses on HumanMelanoma Growth In Vivo

Systemic virotherapy of human melanoma tumors in mice was assessed usingthree different human melanoma cells to establish tumors, and differentviruses for treatment. Human 888-MEL cells, 1858-MEL cells or 1936-MELcells (gift from Dr. F. Marincola at the National Institutes of Health,Bethesda, Md.; see e.g. Wang et al., (2006) J. Invest. Dermatol.126:1372-1377) were implanted subcutaneously into the right lateralthigh of nude mice at a dose of 1×10⁶cells, 4×10⁶ cells and 1×10⁶ cells,respectively, in 100 μl PBS. GLV-1h68, GLV-1h90 or GLV-1h96 at a dose of5×10⁶ PFU in 100 μl PBS were injected i.v. into the femoral vein of micewhen the tumor was established. This corresponded to injection of virus51 days after implantation into mice bearing 888-MEL cells; 27 daysafter implantation into mice bearing 1858-MEL cells; and 72 days afterimplantation in mice bearing 1936-MEL cells. The control groups of micewas not given any treatment. Tumor volume (mm³) was measured atdifferent time points post tumor cell injection. Results of median888-MEL, 1858-MEL and 1936-MEL tumor volume are provided in Tables 49,50 and 51, respectively.

Each virus provided for a decrease in median tumor volume, relative touninfected control mice, in mice bearing 888-MEL tumors (Table 49). Micethat received no treatment were sacrificed due to excessively largetumors reaching a median volume of 2166.8 mm³ at 83 days postimplantation. GLV-1h96, which expresses IL-24, exhibited the best tumortherapy efficacy with a median tumor volume that reached 843.1 mm³ 110days after implantation. GLV-1h68, which does not express aninterleukin, and GLV1h90, which expresses IL-6, exhibited similar tumortherapy efficacy. Median tumor volumes at 110 days post implantationwere 1657.2 mm in mice treated with GLV-1h68, and 1829.4 mm 3 in micetreated with GLV-1h90.

TABLE 49 Median tumor volumes at different time points after i.v.injection of different modified viruses into mice bearing 888-MEL tumorsDays post- implantation Median tumor volume (mm³) of tumor GLV- GLV-GLV- cells No treatment 1h68 1h90 1h96 50 144.75 292.4 153.7 460 57 217463.65 256.9 732.5 63 438.05 667.35 381.2 895.2 66 549.45 559.85 465.8763.2 71 945.55 653.95 446.3 668.6 76 1438.4 712 510 657.2 83 2166.3 669783.2 582.6 91 * 677.4 925 636.9 100 * 926.4 1055.7 698 105 * 1272.951318.9 698.9 111 * 1657.2 1829.4 843.1

The 1858-MEL tumors of mice treated with the different viruses weremarkedly smaller in volume relative to uninfected control mice (Table50). GLV-1 h96 again exhibited the best tumor therapy efficacy with amedian tumor volume that reached only 13.3 mm³ 59 days afterimplantation, which is approximately 14% of the volume of tumors inuntreated mice at the same time point. Treatment with GLV-1h68 alsoslowed tumor growth in mice, compared to untreated mice. By day 59, micetreated with GLV-1h68 had median tumor volumes of 182.4 mm³, compared to900 mm³ in untreated mice. GLV-1h90 exhibited slightly less tumortherapy efficacy compared to the other viruses (median tumor volume of245.65 mm³ at day 59), but still slowed tumor growth compared to notreatment.

TABLE 50 Median tumor volumes at different time points after i.v.injection of different modified viruses into mice bearing 1858-MELtumors Days post- implantation Median tumor volume (mm³) of tumor GLV-GLV- GLV- cells No treatment 1h68 1h90 1h96 27 81.45 108.95 109.9 105.834 148.8 204 161.3 171.6 39 206.05 217.6 182.4 178.2 45 356.9 202.5216.8 171.3 52 695.6 182.9 239.8 148.6 59 900 182.4 245.65 130.3

Tumor therapy efficacy of GLV-1h68 and GLV-1h96 also was assessed inmice bearing 1936-MEL tumors (Table 51). In this experiment, GLV-1h68exhibited the best efficacy, slowing tumor growth by approximately 50%compared to that observed in untreated mice (median tumor volume of1572.6 mm³ at day 118 compared to 3175 mm³). GLV-1h96 also exhibitedsome tumor therapy efficacy, resulting in median tumor volumes of 2878.4mm in mice treated with this virus.

TABLE 51 Median tumor volumes at different time points after i.v.injection of different modified viruses into mice bearing 1936-MELtumors Days post- Median tumor volume (mm³) implantation GLV- GLV- oftumor cells No treatment 1h68 1h96 72 477.7 399.1 650.95 80 581.7 745.8854.9 85 725.1 876.7 1043.6 91 1024.3 1088.8 1147.3 100 1429 1209 1634.1105 1918.15 1290.1 1934.95 111 2664.6 1425.9 2532.65 118 3175 1572.62878.4

Example 32 Effects of different doses of IL-6 and IL-24 ExpressingViruses on the Health of Mice

The effects of different doses of virally expressed IL-6 and IL-24 onthe health of mice was evaluated by assessing body weight. Groups of 4-5week old C57BL/6 mice were injected intravenously [in 100 μl of PBS,through tail vein] with 5×10⁷ PFU, 1×10⁸ PFU or 2×10⁸ PFU of GLV-1h68,GLV-1h90 (expressing IL-6) and GLV-1h96 (expressing IL-24),respectively. Body weight was measured at 30, 36, 45, 52 and 58 dayspost-cancer cell injection. Results of median body weight (gm) areprovided in Table 52. None of the viruses appeared to have a detrimentalaffect on the body weight of the mice.

TABLE 52 Median body weight at different time points after i.v.injection of different modified viruses into mice Days post Median bodyweight (gm) injection GLV-1h68 GLV-1h90 GLV-1h96 of virus 5 × 10⁷ 1 ×10⁸ 2 × 10⁸ 5 × 10⁷ 1 × 10⁸ 2 × 10⁸ 5 × 10⁷ 1 × 10⁸ 2 × 10⁸ 0 17.1 17.617.3 16.6 16.75 16.4 16.5 16.65 15.8 6 18.4 19.4 18.75 18.5 17.95 18.617.8 17.85 18.2 14 19.3 19.4 19.4 19.8 19.2 19.25 18.4 18.25 19.3 2120.4 20.5 21 21 20.45 20.85 20 19.25 20.4 28 21.3 21.1 21.75 20.8 21.6521.8 20.5 20.1 21.7 42 22.4 22.2 22.05 21.6 21.55 22.55 21.1 21 22 5924.3 23 23.7 22.2 22.25 23.1 21.8 24.3

Example 33 Infectivity of GLV-1h68 in Normal and Tumor Fibroblast Cells

The infectivity of GLH-1 h68 in cultures of normal and tumor fibroblastcells was assessed and compared by microscopy and virus titration.Primary human dermal fibroblasts (hDF) were purchased from CellApplications, Inc., and grown in Fibroblast Growth Medium (CellApplication, Inc., San Diego, Calif.). HT-1080 (pLEIN) cells werederived from HT-1080, a human fibrosarcoma cell line (CCL-121, ATCC) bytransfection with a GFP-expressing plasmid. HT-1080 (pLEIN) cells weregrown in DMEM (Mediatech, Inc., Herndon, Va.) with 10% fetal bovineserum (FBS; Mediatech, Inc., Hemdon, Va.). hDF and HT-1080(pLEIN) cells(were seeded at 2×10⁵ cells/well in 24-well plates were infected thefollowing days with a series of 10-fold dilutions of GLV-1h68 induplicate. Two days post infection the plaques were either visualizedunder a fluorescence microscope (Olympus 1X71) using a FITC filter orstained with 0.13% crystal violet to determine the viral titers in bothcell lines. Both the size and number of the plaques in each cell linewas assessed.

The infectivity of GLV-1h68 in human primary dermal fibroblast cells(hDF cells) was approximately 200 times lower than that observed in thehuman fibrosarcoma cell line (HT-1080(pLEIN)). The virus titer from hDFcells was 8.5×10⁶±1.4×10⁶ PFU, compared to 1.7×10⁹±3.5×10⁶ PFU fromHT-1080(pLEIN) cells. GLV-1h68 also was observed to form smaller plaquesin hDF cells compared to HT-1080(pLEIN) cells. These results reflect theselective nature of GLV-1h68 cells for tumor cells versus normal healthycells.

Example 34 Effect of Attenuation of Viruses on Infectivity in PrimaryFibroblast Cells

To investigate the effect of attenuation of viruses on their infectivityin non-tumor cells, viral growth curves of different viruses in aprimary fibroblast cells were determined. Primary murine embryonicfibroblast (MEF) cells were cultured in 12-well plates to 1.1×10⁵cells/well and infected at a multiplicity of infection (MOI) of 0.01with 1×10³ PFU of LIVP, WR, GLV-1d27, GLV-1f65, GLV-1h68, GLV-1h71 anddark 8.1, respectively. After 1 hr at 37° C., the inoculum was aspiratedand the cell monolayers were washed twice with 2 ml of DPBS (Mediatech,Inc., Hemdon, Va.). Two ml of DMEM containing 2% fetal bovine serum(FBS) were added into each well. Three wells of cells from each virusinfection were harvested at 24, 48 and 72 hours post infection. Theharvested cells were subjected to three freeze-thaw cycles and sonicatedthree times for 1 minute at full power before the amount of virus in thelysates was determined by titration. The virus was titrated in CV-1cells in duplicate. Results of the virus titer are provided in Table 53.

Both the LIVP and WR strains established an infection in the primary MEFcells and increased viral titers by 2 to 3 log over 72 hours. Incontrast none of the attenuated viruses (GLV-1d27, GLV-1f65, GLV-1h68,GLV-1h71 or dark 8.1) could replicate in MEF cells, and by 72 hours,none of the attenuated viruses were detectable in cultures of primarymurine embryonic fibroblasts. The loss of infectivity of GLV-1h68 andits parental (GLV-1d27 and GLV-1f65) and derived viruses in non-tumorcells indicates that these viruses have reduced toxicity compared LIVP.This data supports the observations that t in vivo administration ofthese attenuated viruses does not result in viral replication throughoutthe body (see e.g. Example 3), but rather just in tumor cells, therebyreducing in vivo toxicity.

TABLE 53 Virus Replication in Primary Murine Embryonic Fibroblast (MEF)Cells Hours Log Virus Titer (PFU/mL) post GLV- GLV- GLV- GLV- infect.LIVP WR 1d27 1f65 1h68 1h71 dark 8.1 0 3.041 3.041 3.041 3.041 3.0413.041 3.041 24 4.014 4.754 2.009 0.784 0.784 1.091 1.498 48 4.963 5.9470.000 1.133 1.133 0.932 1.780 72 5.292 6.273 0.000 0.000 0.000 0.0000.000

Example 35 Replication of GLV-1h68 in Cat and Dog Tumor Cells

To determine whether virotherapy with GLV-1h68 and its derivatives couldbe used to treat animals other than humans, the replicative ability ofGLV-1h68 in dog and cat tumor cells was assessed.

A. Replication of GLV-1h68 in FC77.T Feline Fibrosarcoma Cells

The ability of GLV-1h68 to replicate in FC77.T feline fibrosarcoma cellsin vitro was investigated. FC77.T feline fibrosarcoma cells (ATCC No.CRL-6105) were cultured in vitro, a process that results in twopopulations of cells: adherent cells and suspension cells in clusters.The culture was infected with 4×10⁶ PFU of GLV-1h68 at an MOI of 0.01,and the infected cells were harvested at 24, 48 and 72 hours postinfection. The amount of virus in cell lysates was determined bytitration of the virus in CV-1 cells. The level of GLV-1h68 infection inthe adherent and suspended cell populations also was monitored bydetection of the fluorescent signal emitted by the virally-encoded GFP.

It was observed in the in vitro GLV-1h68 replication study that cells insuspension displayed green fluorescence, indicating GLV-1h68 replicationin this cell population. In contrast, no fluorescence was observed inadherent cells. Viral titers in the cell lysates of FC77.T felinefibrosarcoma cells dropped from 4×10⁶ PFU of GLV-1h68 to undetectablelevels by 24 hours post infection, indicating that this virus does notreplicate well in these particular feline fibrosarcoma cells.

B. Replication of GLV-1h68 in D17 Dog Osteosarcoma Cells

The ability of GLV-1h68 to replicate in D17 dog osteosarcoma cells (ATCCNo. CCL-183) in vitro, was investigated and compared its replication inGI-101A human breast carcinoma cells. Both cell types were cultured invitro and infected with 4×10⁶ PFU of GLV-1h68 at an MOI of 0.01. Theinfected cells were harvested at 24, 48 and 72 hours post infection andthe amount of virus in cell lysates was determined by titration in CV-1cells. The level of GLV-1h68 infection in the two cell cultures also wasmonitored by detection of the fluorescent signal emitted by thevirally-encoded GFP.

It was observed in the in vitro GLV-1h68 replication study that D17 dogosteosarcoma cells displayed green fluorescence, indicating GLV-1h68replication in this cell population. Viral titration indicated thatGLV-1h68 replication in D17 dog osteosarcoma cells was equally asefficient as that observed in GI-101A human breast carcinoma cells,reaching median log titers of 6.2 in D17 cells compared to 5.9 inGI-101A cells by 72 hours post infection.

These studies in cat and dog tumor cells indicate that GLV-1h68 canreplicate in animal cells other than human cells, and, therefore, thatGLV-1h68 and its derivatives have the potential to be used invirotherapy treatments in animals other than humans.

Example 36 Cellular Immunity to GLV-1h68 in Mice

The ability of GLV-1h68 to induce a cellular immune response in mice wasinvestigated by evaluating the cytotoxic T cell (CTL) response in invitro chromium release CTL assays. Preliminary development experiments(Experiments 1 through 4) using only a few mice each were performed todetermine the appropriate conditions and parameters for a larger study(Experiment 5) involving more animals. A detailed description of themethods for the CTL assay used in these experiments is provided insection 5, below.

1. Experiment 1

To determine the appropriate length of time for infection of targetcells for the CTL assay, mice treated with 5×10⁶ PFU of GLV-1h68 (n=3)on day 1 and 14, by intravenous injection through the tail vein, anduntreated control mice (n=2), were sacrificed on day 21. The effectorcell splenocytes were isolated from the spleens and were mixed with1×10⁴ MC57G target cells (ATCC No. CRL-2295) that had previously beeninfected with GLV-1h68 either for 2 hours (MOI of 5) with concurrent⁵¹Cr labeling, or infected overnight (MOI of 5) followed by a 2 hour⁵¹Cr labeling incubation. The ratios at which the effector and targetcells were mixed for the CTL assay were 3:1, 10:1, 30:1 and 90:1, andthe cells were incubated for 4 hours. Following incubation, the cellswere washed and lysed and the supernatant was assayed for radioactivity(in counts per minute) using a scintillation counter. It was observedthat the CTL response in mice treated twice with GLV-1h68 was strongerwhen the target cells were infected for 2 hours (approximately 45-60%specific lysis at an E:T ratio of 90:1) compared with overnightinfection (approximately 25-35% specific lysis). Splenocytes fromuntreated mice showed negligible specific lysis. These data indicatethat a 2 hour infection with an MOI of 5 is appropriate for use insubsequent experiments.

2. Experiment 2

To assess the effect of restimulation of effector cells in vitro, aportion of the splenocytes from the Experiment I were restimulated invitro by the addition of GLV-1h68 at an MOI of 0.25 for 1 week beforethey were used in a CTL assay with MC57G target cells that had beeninfected for 2 hours with GLV1h68 at an MOI of 5 and labeled with ⁵¹Cr.The CTL response was greater when effector cells were used after a 1week restimulation, compared to no restimulation. Effector cells frommice treated twice with GLV-1h68, and restimulated for 1 week in vitroexhibited 50-70% specific lysis at an E:T ratio of 30:1, compared toapproximately 25% specific lysis using cells that had not beenrestimulated (from Experiment 1). The CTL response from effector cellsfrom untreated mice that had been restimulated for 1 week in vitro alsoincreased, reaching approximately 15% specific lysis at an E:T ratio of30:1. Recovery of cells, however, was poor, which could render thismethod inappropriate for some studies.

3. Experiment 3

To determine the effect of a freeze-thaw cycle on splenocytes prior touse in the CTL assay, splenocytes isolated from mice treated once withGLV-1h68 were frozen immediately on isolation and stored frozen inliquid nitrogen vapor. The cells were then thawed and restimulated withGLV-1h68 at an MOI of 0.25 for 1 week before they were used as effectorsin a CTL assay with MC57G target cells that had been infected for 2hours with GLV1h68 at an MOI of 5 and labeled with ⁵¹Cr. Freezing of thesplenocytes from GLV-1h68 treated mice resulted in specific lysis of upto 100% at an E:T ratio of 60:1. After thawing and 1 week ofrestimulation, however, no splenocytes were recovered from untreatedanimals. Thus, in vitro restimulation was not included in subsequentexperiments.

4. Experiment 4

In a fourth preliminary experiment, splenocytes from animals treatedeither once or twice with GL-ONC1 isolated and subjected to a freezethaw cycle prior to incubation in a CTL assay with MC57G target cellsthat had been infected with GLV-1h68 and labeled with ⁵¹Cr. The CTLresponse after a freeze-thaw cycle (and no in vitro restimulation) wasgreater in mice treated twice with GLV-1h68 (approximately 20% specificlysis at an E:T ratio of 60:1) compared to mice treated only once withGLV-1h68 (less that 5% specific lysis at an E:T ratio of 60:1). Thisdata indicates that it may be more appropriate to test fresh instead offreeze-thawed splenocytes from animals treated once with GLV-1h68.

5. Experiment 5

Experiments 1-4 were used to establish appropriate conditions andparameters for the following larger study of the cellular immuneresponse in mice treated with GLV-1h68. Ten 10 week old C57/BL6 mice (5male and 5 female) were injected intravenously into the tail vein with5×10⁶ PFU of GLV-1h68 in 500 μl PBS. A control group of 10 C57/BL6 mice(5 male and 5 female) were treated with PBS alone. Twenty-one days afterinjection, the mice were sacrificed and the spleens were removedascetically. The excess fat was trimmed off the spleens and the spleenswere transferred to 5 ml tubes containing 2-3 ml sterile culture mediumand stored on ice for transport.

To prepare the effector cells for the CTL assay, single cell suspensionsof the mouse splenocytes were prepared before the cells were filteredthrough a 40-70 μm sterile cell strainer and centrifuged at 250-350×gfor 8 to 10 mins at 15-20° C. The supernatant was aspirated and thecells were resuspended at 9×10⁶ cells/ml in pre-warmed effector medium(RPMI-1640 with 10% heat inactivated FBS, 55 μM 2-mercaptoethanol, 1%GlutaMAX (Invitrogen, CA) and 1 mM sodium pyruvate). The cells werefurther diluted to 3×10⁶ cells/ml, 1×10⁶ cells/ml and 3×10⁵ cells/ml.

To prepare the target cells for the CTL assay, 3×10⁵ cells/ml MC57Gcells (ATCC No. CRL-2295) were cultured in a tissue culture flaskovernight in target medium (EMEM with 10% heat inactivated FBS, 100 μMnon-essential amino acids, 1% GlutaMAX and 1 mM sodium pyruvate). Astock of 1×10⁸ PFU/ml of GLV-1h68 was prepared in target medium, and astock of 1 mCi/ml of ⁵¹Cr also was prepared. The MC57G cells wereharvested and 1.5×10⁶ cells were resuspended in 0.35 ml target medium ina 15 ml tube, to which was added 0.075 ml of ⁵¹Cr and 0.075 ml ofGLV-1h68. Uninfected target cells also were prepared as controls byadding 0.075 ml of ⁵¹Cr and 0.075 ml of target medium (no virus). Thetarget cells were then incubated for 2 hours at 37° C. Followingincubation the cells were washed three times in effector medium andcounted.

The CTL assay was initiated by adding 1×10⁴ target cells (infected oruninfected) in 100 μl to the wells of a 96 well plate. An equal volumeof effector cells were then added to each well such that theeffector:target cell (E:T) ratios were 30:1, 90:1, 270:1 and 540:1.Additional controls to assess maximum lysis and spontaneous release of51 Cr were included in the assay by plating 100 μl target cells with 100μl 2% lysis buffer (2% Triton X-100 in effector medium), and 100 μltarget cells with 100 μl effector medium, respectively. A furtherpositive control using pooled splenocytes (after a freeze-thaw cycle)from mice treated twice (on day I and day 14) with 5×10⁶ PFU GLV-1h68also was included, using E:T ratios of 3.3:1, 10:1, 30:1 and 90:1. The96 well plates were centrifuged at 150×g for 5 minutes and thenincubated at 37° C. for 4 hours. The plates were again centrifuged at150×g for 5 minutes and 50 μl supernatant from each well was added tothe corresponding well of a 96 well plate containing 150 μlscintillation fluid/well. The radioactivity (counts per minute (CPM))was measured using a scintillation counter. The percentage specificlysis of target cells for each effector:target ratio are provided inTable 54. Statistical analysis of the data was performed using a t-test.

The mean percent specific lysis using effector cells from GLV-1h68treated animals was greater than that of mice treated with PBS at eacheffector:target ratio (values ranged from 0.3% to 3.8% in controls and2.1% to 6.4% in GLV-1h68 treated animals). A very strong CTL responsewas elicited in mouse 18, reaching levels approximately twice that ofother mice administered GLV-1h68. Statistical analysis revealed that theincreases in CTL response in mice that received a single GLV-1h68injection compared to untreated mice were statistically significant ateach effector:target ratio (probability value (p)=0.0016 for the E:Tratio of 30:1; p=0.0010 for the E:T ratio of 90:1; p=0.0021 for the E:Tratio of 270:1; and p=0.0315 for the E:T ratio of 540:1). When animal 18(a putative outlier, discussed above) was excluded from analysis, meanpercent specific lysis using effector cells from GLV-1h68 treatedanimals remained greater than that for cells from control animals, butthe differences were statistically significant only at the 30:1, 90:1,and 270:1 effector:target ratio.

As shown in Table 54, there was more robust CTL activity using thepositive control cells, which were obtained from mice treated twice withGLV-1h68. Percent specific lysis values in this cell population rangedfrom 5.0% (3.3:1 effector:target ratio) to 37.0% (90:1 effector:targetratio).

This data indicates that intravenous administration of GLV-1h68 to miceelicits a specific cytotoxic T cell response that can be readilydetected using standard cellular immunity assays.

TABLE 54 Cytotoxic T cell activity in mice treated with GLV-1h68Percentage specific lysis Uninfected Infected target cells target cellsE:T E:T E:T E:T E:T Animal Treatment 30:1 90:1 270:1 540:1 270:1  1 PBS0.2% 0.8% 3.3% 6.2% 3.2%  2 PBS 1.1% 1.1% 3.8% 5.9% 2.6%  3 PBS 0.9%0.7% 2.2% 4.2% 1.4%  4 PBS 0.6% 1.6% 3.3% 4.8% 1.6%  5 PBS 0.5% 0.5%2.2% 4.1% 1.3%  6 PBS 0.0% 1.2% 3.0% 4.8% 1.9%  7 PBS 0.7% 0.9% 3.1%4.5% 2.8%  8 PBS 1.0% −0.1% 0.4% 0.8% 2.3%  9 PBS −0.9% −0.1% 1.4% 1.0%2.6% 10 PBS −0.9% −0.3% 1.3% 1.3% 1.2% Group mean 0.3% 0.6% 2.4% 3.8%2.1% Standard deviation 0.7% 0.6% 1.1% 2.0% 0.7% 11 GLV-1h68 0.3% 1.1%2.9% 3.2% 1.8% 12 GLV-1h68 0.8% 3.1% 4.5% 4.7% 1.9% 13 GLV-1h68 2.2%3.1% 5.2% 4.2% 3.5% 14 GLV-1h68 1.3% 2.9% 8.0% 8.3% 2.5% 15 GLV-1h681.5% 4.5% 5.5% 6.5% 2.6% 16 GLV-1h68 2.0% 3.2% 4.3% 4.4% 1.9% 17GLV-1h68 2.9% 4.8% 7.6% 7.1% 2.7% 18 GLV-1h68 5.1% 11.3% 14.6% 11.7%3.5% 19 GLV-1h68 2.3% 2.6% 4.0% 4.1% 1.7% 20 GLV-1h68 2.6% 4.6% 7.1%7.5% 2.6% Group mean^(a) 2.1% 4.1% 6.4% 6.2% 2.5% Standard deviation^(a)1.3% 2.8% 3.3% 2.6% 0.6% Group mean^(b) 1.8% 3.3% 5.5% 5.6% 2.4%Standard deviation^(b) 0.8% 1.2% 1.8% 1.8% 0.6% E:T E:T E:T E:T E:T3.3:1 10:1 30:1 90:1 290:1 Positive control 5.0% 9.8% 20.0% 37.0% 1.9%^(a)Including all animals (11-20). ^(b)Excluding animal 18. ^(c)Pooledsplenocytes (after freeze-thaw cycle) from animals treated twice withGL-ONC1.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A method for modulating attenuation of a therapeutic virus,comprising: a) providing a therapeutic virus for attenuation modulation,wherein: the therapeutic virus comprises a heterologous nucleic acidcontaining an open reading frame; the open reading frame encodes apolypeptide; and the heterologous nucleic acid is operably linked to apromoter; and b) modifying the heterologous nucleic acid molecule,whereby modification alters attenuation of the resulting virus comparedto the attenuation of the unmodified therapeutic virus.
 2. The method ofclaim 1, wherein the open reading frame encodes a non-therapeuticpolypeptide.
 3. The method of claim 1, wherein the open reading frameencodes a therapeutic polypeptide.
 4. The method of claim 1, furthercomprising: assessing the level of attenuation following modification ofthe virus.
 5. The method of claim 4, wherein the step of assessing thelevel of attenuation is performed in vitro or in vivo.
 6. The method ofclaim 1, wherein the modification increases or decreases the level ofattenuation relative to the unmodified virus.
 7. The method of claim 5,wherein attenuation is assessed by changes in one or more of thefollowing properties of the virus: a) viral mRNA synthesis; b) viralprotein expression; c) viral DNA replication; d) viral plaque size; e)viral titer; and/or f) in vivo toxicity.
 8. The method of claim 1,wherein the modification comprises replacement or removal of all or aportion of the heterologous nucleic acid molecule, wherein the removalalters the attenuation of the virus.
 9. The method of claim 8, whereinthe portion of the heterologous nucleic acid that is replaced or removedcomprises 1, 2, 3, 4, 5 or more, 10 or more, 15 or more, 20 or more, 50or more, 100 or more, 1000 or more, 5000 or more nucleotide bases. 10.The method of claim 8, wherein modification comprises replacement of allor a portion of the heterologous nucleic acid molecule with a non-codingnucleic acid molecule.
 11. The method of claim 1, wherein the promoterthat is operably linked to the heterologous nucleic acid is a nativepromoter or a heterologous promoter.
 12. The method of claim 11, whereinthe promoter is a synthetic promoter.
 13. The method of claim 11,wherein the promoter is a viral promoter.
 14. The method of claim 13,wherein the promoter is a poxvirus promoter.
 15. The method of claim 14,wherein the promoter is a vaccinia viral promoter.
 16. The method ofclaim 15, wherein the promoter is selected from among a vaccinia early,intermediate, early/late and late promoter.
 17. The method of claim 16,wherein the promoter is selected from among vaccinia P_(7.5k), P_(11k),P_(EL), P_(SEL), P_(SE), H5R, TK, P28, C11R, G8R, F17R, I3L, I8R, A1L,A2L, A3L, H1L, H3L, H5L, H6R, H8R, D1R, D4R, D5R, D9R, D11L, D12L, D13L,M1L, N2L, P4b or K1.
 18. The method of claim 13, wherein the promoter isselected from among an adenovirus late promoter, Cowpox ATI promoter, orT7 promoter.
 19. The method of claim 1, wherein the promoter that isoperably linked to the heterologous nucleic acid is modified.
 20. Themethod of claim 19, wherein the modification comprises replacement ofthe promoter with another promoter, wherein: the replaced promoter isstronger thereby resulting in increased attenuation; or the replacedpromoter is weaker, thereby resulting in decreased attenuation.
 21. Themethod of claim 20, wherein the replaced promoter is selected from amongvaccinia P_(7.5k), P_(11k), P_(EL), P_(SEL), P_(SE), H5R, TK, P28, C11R,G8R, F17R, I3L, I8R, A1L, A2L, A3L, H1L, H3L, H5L, H6R, H8R, D1R, D4R,D5R, D9R, D11L, D12L, D13L, M1L, N2L, P4b or K1.
 22. The method of claim1, wherein the open reading frame encodes one or more gene products. 23.The method of claim 1, wherein the open reading frame is modified,wherein the modification alters the attenuation of the virus compared tothe attenuation of the unmodified virus.
 24. The method of claim 23,wherein the modification comprises increasing the length of the openreading frame or removal of all or part of the open reading frame,thereby increasing the level of attenuation.
 25. The method of claim 1,wherein the modification comprises replacement of all or a portion of afirst heterologous nucleic acid molecule with a second heterologousnucleic acid molecule comprising an open reading frame operably linkedto a promoter, wherein replacement alters attenuation of the resultingvirus compared to the attenuation of the unmodified virus.
 26. Themethod of claim 25, wherein the second heterologous nucleic acidmolecule comprises: a stronger promoter, thereby resulting in increasedattenuation; or a weaker promoter, thereby resulting in decreasedattenuation.
 27. The method of claim 25, wherein the second heterologousnucleic acid molecule comprises two or more promoters and/or two or moreopen reading frames.
 28. The method of claim 1, wherein modificationalters transcription and/translation of one or more viral genes duringinfection.
 29. The method of claim 1, wherein modification alterstranslation of one or more endogenous viral polypeptides duringinfection.
 30. The method of claim 1, further comprising insertion ofone or more heterologous nucleic acid molecules comprising an openreading frame operably linked to a promoter into the viral genome. 31.The method of claim 1, wherein the modified virus encodes a detectableprotein or a protein that induces a detectable signal.
 32. The method ofclaim 31, wherein the protein is selected from a luciferase, afluorescent protein or a protein that binds a contrasting agent,chromophore, or a compound or detectable ligand.
 33. The method of claim32, wherein the protein is a fluorescent protein that is a greenfluorescent protein or red fluorescent protein.
 34. The method of claim1, further comprising insertion of a heterologous nucleic acid moleculethat encodes a therapeutic gene product.
 35. The method of claim 34,wherein the therapeutic gene product is selected from among a cytokine,a chemokine, an immunomodulatory molecule, a single chain antibody,antisense RNA, siRNA, prodrug converting enzyme, a toxin, a mitosisinhibitor protein, an antitumor oligopeptide, an anti-cancer polypeptideantibiotic, angiogenesis inhibitor, or tissue factor.
 36. The method ofclaim 1, wherein the virus is selected from among a poxvirus,herpesvirus, adenovirus, adeno-associated virus, lentivirus, retrovirus,rhabdovirus and a papillomavirus.
 37. The method of claim 1, wherein thevirus is a vaccinia virus.
 38. The method of claim 37, wherein the virusis a vaccinia virus that is a Lister strain.
 39. The method of claim 38,wherein the Lister strain is LIVP.
 40. The method of claim 38, whereinthe vaccinia virus is selected from among GLV-1h22, GLV-1h68, GLV-1i69,GLV-1h70, GLV-1h71, GLV-1h72, GLV-1 h73, GLV-1h75, GLV-1h81, GLV-1h82,GLV-1h83, GLV-1h84, GLV-1h85, GLV-1h86, GLV-1j87, GLV-1j88, GLV-1j89,GLV-1h90, GLV-1h91, GLV-1h92, GLV-1 h96, GLV-1h97, GLV-1h98, GLV-1h104,GLV-1h105, GLV-1h106, GLV-1h107, GLV-1h108 and GLV-1h109.
 41. The methodof claim 1, further comprising determining the desired level ofattenuation, wherein the desired level of attenuation depends on thetherapeutic or diagnostic application of the virus.
 42. The method ofclaim 41, wherein the application is a therapeutic application thatcomprises treatment of a tumor, cancer or metastasis.
 43. The method ofclaim 41, wherein the application is a diagnostic application thatcomprises detection of a tumor.
 44. The method of claim 41, whereindetermining the desired level of attenuation comprises assessing thehealth of a subject prior to administration of the virus to the subject.45. The method of claim 41, wherein the desired level of attenuationdepends on the route of administration for the virus for the diagnosticor therapeutic application.